Modifications for antisense compounds

ABSTRACT

The invention pertains to modifications for antisense oligonucleotides, wherein the modifications are used to improve stability and provide protection from nuclease degradation. The modifications could also be incorporated into double-stranded nucleic acids, such as synthetic siRNAs and miRNAs.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.13/227,286, filed Sep. 7, 2011, which claims the benefit of priorityfrom U.S. Provisional Application No. 61/380,586, filed Sep. 7, 2010,the disclosures of which are incorporated by reference herein in theirentireties. This application is also a continuation-in-part of U.S.application Ser. No. 13/073,866, filed Mar. 28, 2011, which claims thebenefit of priority from U.S. Provisional Application No. 61/318,043,filed Mar. 26, 2010.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Small BusinessInnovation Research (SBIR) Grant No. GM085863 awarded by the NationalInstitute of General Medical Sciences of the National Institutes ofHealth (NIH). The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention pertains to modifications for antisense oligonucleotides,wherein the modifications are used to improve binding affinity andprovide protection from nuclease degradation.

BACKGROUND OF THE INVENTION

Antisense oligonucleotides (ASOs) are synthetic nucleic acids that bindto a complementary target and suppress function of that target.Typically ASOs are used to reduce or alter expression of RNA targets,particularly messenger RNA (mRNA) or microRNA (miRNA) species. As ageneral principle, ASOs can suppress gene expression via two differentmechanisms of action: 1) by steric blocking, wherein the ASO tightlybinds the target nucleic acid and inactivates that species, preventingits participation in cellular activities, or 2) by triggeringdegradation, wherein the ASO binds the target and leads to activation ofa cellular nuclease that degrades the targeted nucleic acid species. Oneclass of “target degrading” ASOs is “RNase H active”, where formation ofheteroduplex nucleic acids by hybridization of the target RNA with aDNA-containing RNase H active ASO forms a substrate for the enzyme RNaseH. RNase H degrades the RNA portion of the heteroduplex molecule,thereby reducing expression of that species. Degradation of the targetRNA releases the ASO, which is not degraded, and is then free to recycleand bind another RNA target of the same sequence. For an overview ofantisense strategies, oligonucleotide design and chemical modifications,see Kurreck, 2003, Eur. J. Biochem., 270(8): 1628-44.

Unmodified DNA oligonucleotides have a half-life of minutes whenincubated in human serum. Therefore, unmodified DNA oligonucleotideshave limited utility as ASOs. The primary nuclease present in serum hasa 3′-exonuclease activity (Eder et al., 1991, Antisense Res. Dev. 1(2):141-51). Once an ASO gains access to the intracellular compartment, itis susceptible to endonuclease degradation. Historically, the firstfunctional ASOs to gain widespread use comprised DNA modified withphosphorothioate groups (PS). PS modification of internucleotidelinkages confers nuclease resistance, making the ASOs more stable bothin serum and in cells. As an added benefit, the PS modification alsoincreases binding of the ASO to serum proteins, such as albumin, whichdecreases the rate of renal excretion following intravenous injection,thereby improving pharmacokinetics and improving functional performance(Geary et al., 2001, Curr. Opin. Investig. Drugs, 2(4): 562-73).However, PS-modified ASOs are limited to a 1-3 day half-life in tissue,and the PS modifications reduce the binding affinity of the ASO for thetarget RNA, which can decrease potency (Stein et al., 1988, NucleicAcids Res. 16(8): 3209-21).

The PS modification is unique in that it confers nuclease stability, yetstill permits formation of a heteroduplex with RNA that is a substratefor RNase H. Most other modifications that confer nuclease resistance,such as methyl phosphonates or phosphoramidates, are modifications thatdo not form heteroduplexes that are RNase H substrates when hybridizedto a target mRNA. Improved potency could be obtained using compoundsthat were both nuclease resistant and showed higher affinity to thetarget RNA, yet retain the ability to activate RNase H mediateddegradation pathways.

Further design improvements were implemented to increase affinity forthe target RNA while still maintaining nuclease resistance (see Walderet al., U.S. Pat. No. 6,197,944 for designs containing 3′-modificationswith a region containing unmodified residues with phosphodiesterlinkages; see also European Patent No. 0618925 for “Gapmer” compoundshaving 2′-methoxyethylriboses (MOE's) providing 2′-modified “wings” atthe 3′ and 5′ ends flanking a central 2′-deoxy gap region). This newstrategy allows for chimeric molecules that have distinct functionaldomains. For example, a single ASO can contain a domain that confersboth increased nuclease stability and increased binding affinity, butitself does not form an RNase H active substrate; a second domain in thesame ASO can be RNase H activating. Having both functional domains in asingle molecule improves performance and functional potency in antisenseapplications. One successful strategy is to build the ASO from differentchemical groups, with a domain on each end intended to confer increasedbinding affinity and increased nuclease resistance, each flanking acentral domain comprising different modifications. This facilitatesRNase H activation. This so-called “end blocked” or “gapmer” design isthe basis for the improved function “second generation” ASOs. Compoundsof this design are typically significantly more potent as gene knockdownagents than the “first generation” PS-DNA ASOs.

Typically ASOs that function using steric blocking mechanisms of actionshow higher potency when made to maximize binding affinity. This can beaccomplished through use of chemical modifications that increase bindingaffinity, such as many of the 2′-ribose modifications discussed herein,minor groove binders, or the internal non-base modifiers of the presentinvention. Alternatively, increased binding affinity can be achieved byusing longer sequences. However, some targets are short, such as miRNAs,which are typically only 20-24 bases long. In this case, making ASOslonger to increase binding affinity is not possible. Furthermore, shortsynthetic oligonucleotides gain access into cells more efficiently thanlong oligonucleotides, making it desirable to employ short sequenceswith modifications that increase binding affinity (see, e.g., Straarupet al., 2010, Nucleic Acids Res. 38(20): 7100-11). The chemicalmodification and methods of the present invention enable synthesis ofrelatively short ASOs having increased binding affinity that showimproved functional performance.

ASO modifications that improve both binding affinity and nucleaseresistance typically are modified nucleosides that are costly tomanufacture. Examples of modified nucleosides include locked nucleicacids (LNA), wherein a methyl bridge connects the 2′-oxygen and the4′-carbon, locking the ribose in an A-form conformation; variations ofLNA are also available, such as ethylene-bridged nucleic acids (ENA)that contain an additional methyl group, amino-LNA and thio-LNA.Additionally, other 2′-modifications, such as 2′-O-methoxyethyl (MOE) or2′-fluoro (2′-F), can also be incorporated into ASOs. Some modificationsdecrease stability, and some can have negative effects such as toxicity(see Swayze et al., 2007, Nucleic Acids Res. 35(2): 687-700).

The present invention provides for non-nucleotide modifying groups thatcan be inserted between bases in an ASO to improve nuclease resistanceand binding affinity, thereby increasing potency. The novelmodifications of the present invention can be employed with previouslydescribed chemical modifications (such as PS internucleotide linkages,LNA bases, MOE bases, etc.) and with naturally occurring nucleic acidbuilding blocks, such as DNA or 2′-O-Methyl RNA (2′OMe), which areinexpensive and non-toxic. These and other advantages of the invention,as well as additional inventive features, will be apparent from thedescription of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides non-nucleotide modifications for antisenseoligonucleotides, wherein the modifications are used to increase bindingaffinity and provide protection from nuclease degradation.

The invention also provides an antisense oligonucleotide comprising atleast one modification that is incorporated at the terminal end of anantisense oligonucleotide, or between two bases of the antisenseoligonucleotide, wherein the modification increases binding affinity andnuclease resistance of the antisense oligonucleotide. In one embodiment,the antisense oligonucleotide comprises at least one modification thatis located within three bases of a terminal nucleotide. In anotherembodiment, the antisense oligonucleotide comprises at least onemodification that is located between a terminal base and a penultimatebase of either the 3′- or the 5′-end of the oligonucleotide. In anotherembodiment, the antisense oligonucleotide comprises a modification at aterminal end of the oligonucleotide. In a further embodiment, theantisense oligonucleotide comprises a modification at the terminal endor between the terminal base and the penultimate base of both the 3′-and the 5′-ends of the antisense oligonucleotide. In yet a furtherembodiment, the oligonucleotide contains a non-base modifier at aterminal end or between the terminal base and the penultimate base atthe 5′-end and at the 3′-end. The relative increase of binding affinitycontributed by the non-base modifier may vary with sequence context(Example 8) which can influence which of the various design optionstaught herein is most potent.

The invention further provides an antisense oligonucleotide comprisingat least one modification that is incorporated at the terminal end orbetween two bases of the antisense oligonucleotide, wherein themodification increases binding affinity and nuclease resistance of theantisense oligonucleotide, and wherein the modification is a napthyl-azocompound.

The invention further provides an antisense oligonucleotide comprisingat least one modification that is incorporated at a terminal end orbetween two bases of the antisense oligonucleotide, wherein themodification increases binding affinity and nuclease resistance of theantisense oligonucleotide, and wherein the modification has thestructure:

wherein the linking groups L₁ and L₂ positioning the modification at aninternal position of the oligonucleotide are independently an alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R₁-R₅ areindependently a hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl,substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl,alkylaryl, alkoxy, an electron withdrawing group, an electron donatinggroup, or an attachment point for a ligand; and X is a nitrogen orcarbon atom, wherein if X is a carbon atom, the fourth substituentattached to the carbon atom can be hydrogen or a C1-C8 alkyl group.

The invention further provides an antisense oligonucleotide comprisingat least one modification that is incorporated at a terminal end orbetween two bases of the antisense oligonucleotide, wherein themodification increases binding affinity and nuclease resistance of theantisense oligonucleotide, and wherein the modification has thestructure:

wherein the linking groups L₁ and L₂ positioning the modification at aninternal position of the oligonucleotide are independently an alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R₁, R₂, R₄,R₅ are independently a hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl,substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl,alkylaryl, alkoxy, an electron withdrawing group, or an electrondonating group; R₆, R₇, R₉-R₁₂ are independently a hydrogen, alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron withdrawinggroup, or an electron donating group; R₈ is a hydrogen, alkyl, alkynyl,alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substitutedaryl, cycloalkyl, alkylaryl, alkoxy, or an electron withdrawing group;and X is a nitrogen or carbon atom, wherein if X is a carbon atom, thefourth substituent attached to the carbon atom can be hydrogen or aC1-C8 alkyl group. In one embodiment, R₈ is NO₂.

The invention further provides an antisense oligonucleotide comprisingat least one modification that is incorporated at a terminal end orbetween two bases of the antisense oligonucleotide, wherein themodification increases binding affinity and nuclease resistance of theantisense oligonucleotide, and wherein the modification has thestructure:

The antisense oligonucleotides of the invention can include natural,non-natural, or modified bases known in the art. The antisenseoligonucleotides of the invention can also include, typically but notnecessarily on the 3′ or 5′ ends of the oligonucleotide, additionalmodifications such as minor groove binders, spacers, labels, or othernon-base entities. In one embodiment, the antisense oligonucleotidefurther comprises 2′-O-methyl RNA, and optionally comprises at least onenapthyl-azo compound. In another embodiment, the antisenseoligonucleotide further comprises phosphorothioate linkages. In afurther embodiment, the antisense oligonucleotide comprises a region ofbases linked through phosphodiester bonds, wherein the region is flankedat one or both ends by regions containing phosphorothioate linkages.

The invention further provides an antisense oligonucleotide having thestructure:5′-X₁—Z_(n)—X₂—X₃—X₄—Z_(n)—X₅-3′  Formula 4wherein X₁ and X₅ are independently 0-3 nucleotides wherein theinternucleotide linkages are optionally phosphorothioate; wherein Z is anapthyl-azo compound; n is 0 or 1; X₂ and X₄ are independently 1-5nucleotides wherein the internucleotide linkages are optionallyphosphorothioate; and X₃ is 10-25 nucleotides.

In one embodiment, a third modification can be inserted around themiddle of the antisense oligonucleotide. For longer nucleotides (greaterthan 25 bases), additional modifications could be used at intervals toconfer greater stability. In the modifications of the invention, amodifying group is inserted between adjacent bases, thereby generatingan ASO with reduced toxicity and improved affinity and stability. Thebases can be DNA, 2′OMe RNA, or other modified bases. However, modifiedbases do not need to be employed. Because the modifications are insertedbetween the bases, they can be added as phosphoramidite compounds usingstandard phosphoramidite synthesis chemistry.

In a further aspect, the invention provides a method of reducing a levelof a target mRNA in a cell, said method comprising contacting the cellwith an oligonucleotide, wherein said oligonucleotide is at leastpartially complementary to the target mRNA and wherein saidoligonucleotide comprises at least one modification that is incorporatedat the terminal end or between two bases of the oligonucleotide andwherein the modification increases binding affinity and nucleaseresistance of the antisense oligonucleotide, in an amount sufficient toreduce the target mRNA.

In an additional aspect, the invention provides a method of reducing alevel of a target miRNA in a cell, said method comprising contacting thecell with an oligonucleotide, wherein said oligonucleotide is at leastpartially complementary to the target miRNA and wherein saidoligonucleotide comprises at least one modification that is incorporatedat the terminal end or between two bases of the oligonucleotide andwherein the modification increases binding affinity and nucleaseresistance of the antisense oligonucleotide, in an amount sufficient toreduce the target miRNA.

In another aspect the invention provides an oligonucleotidecomplementary to a target mRNA comprising: a modified 3′-terminalinternucleotide phosphodiester linkage, which modified 3′-terminalinternucleotide phosphodiester linkage is resistant to 3′ to 5′exonuclease degradation; modifications on the 3′-terminus and the5′-terminus of the oligonucleotide, wherein the modifications increasebinding affinity of the oligonucleotide to the target mRNA; one or moreadditional modifications, which additional modification(s) facilitate(s)intracellular transport of said oligodeoxynucleotide; and a continuousstretch of at least five nucleotide residues having four internucleotidephosphodiester linkages which are unmodified, wherein saidoligodeoxynucleotide, when mixed with an RNA molecule for which it hascomplementarity under conditions in which an RNaseH is active,hybridizes to the RNA and forms a substrate that can be cleaved by theRNase H.

In one aspect, the invention provides a Dicer-substrate RNA (DsiRNA)oligonucleotide, comprising a sense strand and an antisense strand,wherein at least one modification on the antisense strand isincorporated near the 3′ terminal end or between two bases of theantisense strand.

In a certain aspect, the invention provides an anti-miRNAoligonucleotide (AMO) comprising,

(a) at least one 2′-O-methyl RNA (2′OMe), and

(b) at least one napthyl-azo compound modification that is incorporatedat the terminal end of the AMO, wherein the modification increasesstability of the AMO.

In a further aspect, the invention provides an RNase H antisenseoligonucleotide (ASO) comprising, at least one napthyl-azo compoundmodification that is incorporated at the terminal end of the ASO,wherein each nucleotide is connected by a phosphorothioate group (PS)and wherein the modification increases stability of the ASO.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gel photograph that illustrates the levels of degradation ofsynthetic DNA oligomers in fetal bovine serum. A series of 10-mersingle-stranded DNA oligonucleotides were trace labeled with ³²P attheir 5′-ends and were incubated in serum at 37° C. for the indicatedtimes (0-240 minutes). Reaction products were separated bypolyacrylamide gel electrophoresis (PAGE) and visualized byphosphorimaging. Samples are identified in Table 4.

FIG. 2 illustrates relative miR-21 suppression by various anti-miRNAoligonucleotides (AMOs) using a luciferase reporter assay. A reporterplasmid that expresses both Renilla luciferase and firefly luciferasewas transfected into HeLa cells. Cell extracts were studied for relativeactivity of both enzymes and Renilla luciferase activity was normalizedto firefly luciferase activity. The Renilla luciferase gene contains amiR-21 binding site and miR-21 is highly expressed in HeLa cells.Different anti-miR-21 oligonucleotides (X-axis) were transfected intothe cells and the relative ability of different designs to suppressmiR-21 activity directly relate to the increase in Renilla luciferaseactivity (Y-axis).

FIG. 3 illustrates relative miR-21 suppression by various AMOs using aluciferase reporter assay comparing perfect match and compounds having1, 2, or 3 mismatches (mismatch pattern 1). A reporter plasmid thatexpresses both Renilla luciferase and firefly luciferase was transfectedinto HeLa cells. Cell extracts were studied for relative activity ofboth enzymes and Renilla luciferase activity was normalized to fireflyluciferase activity. The Renilla luciferase gene contains a miR-21binding site and miR-21 is highly expressed in HeLa cells. Differentanti-miR-21 oligonucleotides (X-axis) were transfected into the cells,and the relative ability of different designs to suppress miR-21activity directly relate to the increase in Renilla luciferase activity(Y-axis).

FIG. 4 illustrates relative miR-21 suppression by various AMOs using aluciferase reporter assay comparing perfect match and compounds having1, 2, or 3 mismatches (mismatch pattern 2). A reporter plasmid thatexpresses both Renilla luciferase and firefly luciferase was transfectedinto HeLa cells. Cell extracts were studied for relative activity ofboth enzymes, and Renilla luciferase activity was normalized to fireflyluciferase activity. The Renilla luciferase gene contains a miR-21binding site and miR-21 is highly expressed in HeLa cells. Differentanti-miR-21 oligonucleotides (X-axis) were transfected into the cells,and the relative ability of different designs to suppress miR-21activity directly relate to the increase in Renilla luciferase activity(Y-axis).

FIG. 5 illustrates knockdown of HPRT expression by DNA ASOs, with orwithout PS bonds or iFQ modification. ASOs were transfected into HeLacells and RNA was prepared 24 hours post transfection. Relative HPRTlevels were assessed by RT-qPCR and are reported on the Y-axis.

FIG. 6 illustrates knockdown of HPRT expression by chimeric “gapmer”ASOs, with or without PS bonds and with or without iFQ modification.ASOs were transfected into HeLa cells and RNA was prepared 24 hours posttransfection. Relative HPRT levels were assessed by RT-qPCR and arereported on the Y-axis.

FIG. 7 illustrates knockdown of HPRT using DsiRNAs at doses ranging from0.01 nM to 1.0 nM. DsiRNAs were modified with iFQ group(s) at positionswithin the duplexes as indicated in the schematic below the graph.DsiRNAs were transfected into HeLa cells and RNA was prepared 24 hourspost transfection. Relative HPRT levels were assessed by RT-qPCR and arereported on the Y-axis.

FIG. 8 illustrates the toxicity profiles of various AMO chemistries whentransfected for 24 hours at 50 nM or 100 nM in HeLa cells. The negativecontrol or “NC1” sequence is not predicted to target any known humanmiRNAs or mRNAs, and so toxicity effects should be specific to thechemical composition of the oligonucleotide. The MultiTox-Glo MultiplexCytotoxicity Assay was employed to measure cell viability followingtreatment with various chemically modified oligonucleotides (X-axis),and cell viability was calculated as a ratio of live/dead cells tonormalize the data independent of cell number (Y-axis). A decrease oflive/dead cell values correlates with decreased cell viability.

FIG. 9 illustrates apoptosis induction profiles caused by various AMOchemistries when transfected for 24 hours at 50 nM or 100 nM in HeLacells. The negative control or “NC1” sequence is not predicted to targetany known human miRNAs or mRNAs, and so induction of apoptosis should bespecific to the biological effects of chemical composition of theoligonucleotide in the cell. The Caspase-Glo 3/7 Assay was employed tomeasure the levels of caspase-3 and caspase-7, which are known effectorsof apoptosis, using a luciferase assay. Apoptosis induction followingtreatment with various chemically modified oligonucleotides (X-axis) isproportional to increasing levels of luminescence (Y-axis).

FIG. 10 is a gel photograph that illustrates the levels of degradationof synthetic 2′OMe-RNA oligomers in fetal bovine serum. A series of22-mer single-stranded 2′OMe-RNA oligonucleotides were incubated in 10%serum at 37° C. for the indicated times (0-24 hours). Reaction productswere separated by polyacrylamide gel electrophoresis (PAGE), stainedwith methylene blue, and visualized by transillumination. Samples areidentified in Table 14.

FIG. 11 is a gel photograph that illustrates the levels of degradationof synthetic 2′OMe-RNA oligomers in fetal bovine serum. A series of22-mer single-stranded 2′OMe-RNA oligonucleotides were incubated in 10%serum at 37° C. for the indicated times (0-24 hours). Reaction productswere separated by polyacrylamide gel electrophoresis (PAGE), stainedwith methylene blue, and visualized by transillumination. Samples areidentified in Table 14.

FIG. 12 is a gel photograph that illustrates the levels of degradationof synthetic 2′OMe-RNA oligomers in cell extracts. A series of 22-mersingle-stranded 2′OMe-RNA oligonucleotides were incubated in mouse livercell extracts at 37° C. for the indicated times (0-24 hours). Reactionproducts were separated by polyacrylamide gel electrophoresis (PAGE),stained with methylene blue, and visualized by transillumination.Samples are identified in Table 14.

FIG. 13 is a gel photograph that illustrates the levels of degradationof synthetic 2′OMe-RNA oligomers in cell extracts. A series of 22-mersingle-stranded 2′OMe-RNA oligonucleotides were incubated in mouse livercell extracts at 37° C. for the indicated times (0-24 hours). Reactionproducts were separated by polyacrylamide gel electrophoresis (PAGE),stained with methylene blue, and visualized by transillumination.Samples are identified in Table 14.

FIG. 14 is a gel photograph that illustrates the levels of degradationof synthetic 2′OMe-RNA oligomers in cell extracts. A series of 22-mersingle-stranded 2′OMe-RNA oligonucleotides were incubated in mouse livercell extracts at 37° C. for the indicated times (0-96 hours). Reactionproducts were separated by polyacrylamide gel electrophoresis (PAGE),stained with methylene blue, and visualized by transillumination.Samples are identified in Table 15.

FIG. 15 illustrates relative miR-21 suppression by various AMOs using aluciferase reporter assay. A reporter plasmid that expresses bothRenilla luciferase and firefly luciferase was transfected into HeLacells. Cell extracts were studied for relative activity of both enzymesand Renilla luciferase activity was normalized to firefly luciferaseactivity. The Renilla luciferase gene contains a miR-21 binding site andmiR-21 is highly expressed in HeLa cells. Different anti-miR-21oligonucleotides (X-axis) were transfected into the cells and therelative ability of different designs to suppress miR-21 activitydirectly relate to the increase in Renilla luciferase activity (Y-axis).Sequences are identified in Table 16.

FIG. 16 illustrates relative miR-21 suppression by various AMOs using aluciferase reporter assay. A reporter plasmid that expresses bothRenilla luciferase and firefly luciferase was transfected into HeLacells. Cell extracts were studied for relative activity of both enzymesand Renilla luciferase activity was normalized to firefly luciferaseactivity. The Renilla luciferase gene contains a miR-21 binding site andmiR-21 is highly expressed in HeLa cells. Different anti-miR-21oligonucleotides (X-axis) having various 3′-end structures weretransfected into the cells and the relative ability of different designsto suppress miR-21 activity directly relate to the increase in Renillaluciferase activity (Y-axis). Sequences are identified in Table 18. Thelength of the AMO as synthesized is shown in the name under which isindicated the expected length of the AMO that survives incubation inserum or exposure to cellular nucleases.

FIG. 17 illustrates knockdown of HPRT expression by chimeric “gapmer”ASOs, with or without PS bonds in the 2′-modified flanking domains andwithout further modification, with the FQ modification placed internallynear the ends between the last and penultimate base, or with the FQmodification based at the 5′- and 3′-ends. ASOs were transfected intoHeLa cells and RNA was prepared 24 hours post transfection. RelativeHPRT levels were assessed by RT-qPCR and are reported on the Y-axis.

DETAILED DESCRIPTION OF THE INVENTION

The antisense oligonucleotides of the invention have modificationsplaced at the 3′-end and/or 5′-end, or placed between nucleotides,wherein the modifications increase affinity to the complementary targetand provide nuclease resistance. In one embodiment of the invention, thecompounds are the same as those described in U.S. application Ser. No.13/073,866, the disclosure of which is incorporated by reference hereinin its entirety.

In another embodiment of the invention, the antisense oligonucleotidecomprises at least one modification that has the structure:

wherein the linking groups L₁ and L₂ positioning the modification at aninternal position of the oligonucleotide are independently an alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R₁-R₅ areindependently a hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl,substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl,alkylaryl, alkoxy, an electron withdrawing group, an electron donatinggroup, or an attachment point for a ligand; and X is a nitrogen orcarbon atom, wherein if X is a carbon atom, the fourth substituentattached to the carbon atom can be hydrogen or a C1-C8 alkyl group. In afurther embodiment of the invention, the antisense oligonucleotidecomprises at least one modification that has the structure:

wherein the linking groups L₁ and L₂ positioning the modification at aninternal position of the oligonucleotide are independently an alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R₁, R₂, R₄,R₅ are independently a hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl,substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl,alkylaryl, alkoxy, an electron withdrawing group, or an electrondonating group; R₆, R₇, R₉-R₁₂ are independently a hydrogen, alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron withdrawinggroup, or an electron donating group; R₈ is a hydrogen, alkyl, alkynyl,alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substitutedaryl, cycloalkyl, alkylaryl, alkoxy, or an electron withdrawing group;and X is a nitrogen or carbon atom, wherein if X is a carbon atom, thefourth substituent attached to the carbon atom can be hydrogen or aC1-C8 alkyl group.

The compositions and methods of the invention involve modification of anoligonucleotide by placing non-base modifying group(s) as insertions ata terminal end or between bases while retaining the ability of thatsequence to hybridize to a complementary sequence. Typically, insertionof non-base modifying groups between bases results in a significant lossof affinity of the modified sequence to its complement. The uniquecompositions of the invention, whether the non-base group is located atthe 5′- or 3′-end or internally, increase affinity of the modifiedsequence to its complement, increasing stability and increasing T_(m).Placement of such non-base modifying group(s) prevents nucleases frominitiating degradation at the modified linkage(s). When placedterminally or between the first and second bases at both ends of theoligonucleotide, the sequence is protected from attack by both5′-exonucleases and 3′-exonucleases. Placement at central position(s)within the sequence can additionally confer some resistance toendonucleases. In particular, compounds of the class of Formula 2 aboveimpede nuclease attack for several flanking internucleotide phosphatebonds adjacent to the modified linkage, creating a protected “zone”where unmodified linkages are less susceptible to nuclease cleavage. Themodifications also reduce the rate of cleavage or totally preventcleavage of terminal bases. Thus the compositions and methods of theinvention permit synthesis of ASOs having increased T_(m) and increasednuclease resistance yet do not employ modified but instead employ anon-base modifying group inserted between residues. The non-basemodifier can also be placed directly at the 5′-end and/or 3′-end of theoligomer which will similarly protect the compound from exonucleaseattack.

The ability of the modifying groups of the present invention to increasebinding affinity (T_(m)) of duplexed nucleic acids is demonstrated inExample 1, where melting studies were conducted for a series ofunmodified and modified 10-mer duplex DNA oligomers. Using compositionsand methods of the present invention, an increase of +11° C. wasachieved using only two modifying groups (between the two terminal baseson each end of the oligomer). Similar duplexes made with insertions of apropanediol group show significant destabilization, consistent with theexpected results for non-base insertions. The ability of the modifyinggroups of the present invention to increase binding affinity (T_(m)) ofduplexed nucleic acids is further demonstrated in Examples 8 and 10,where melting studies were conducted for a series of unmodified andmodified 22-mer 2′OMe-RNA oligomers duplexed with an RNA target. Thisexperiment simulates binding of an anti-miRNA antisense oligomer (AMO)with a target miRNA (for Example 10, the target was a synthetic miR-21).In this context, an increase as high as of +4° C. was seen for use ofeven a single modifier. The magnitude of the effect varies with sequenceand in some base contexts was slightly destabilizing.

The ability of the modifying groups of the present invention to improvenuclease stability is demonstrated in Example 2, where single-strandedDNA oligomers were incubated in serum (subjected to degradation by serumnucleases) and then examined for integrity by polyacrylamide gelelectrophoresis (PAGE). Unmodified DNA oligomers are rapidly degraded inserum whereas a 10-mer DNA oligonucleotide with an insertion of thenapthyl-azo modifier between the terminal bases on each end resulted ina compound that was not degraded after 4 hours incubation. Othermodifying groups, such as a propanediol spacer, only slowed the rate ofdegradation slightly. T_(m)-enhancing, nuclease blocking modifications(such as the napthyl-azo group) can be inserted into single-strandedoligomers to improve properties.

Stabilized, increased binding affinity oligomers of this type can have avariety of uses, as is well appreciated by those with skill in the art.As examples (not meant to be limiting), such oligomers can be used asASOs to promote reduction of mRNA or miRNA levels in a cell or animal.Such examples are demonstrated in Examples 3, 4, 5 and 10 below. Thepresent invention can be equally well applied to ASOs intended todegrade a target mRNA or to inhibit function by tight binding (stericblocking) Examples of steric blocking acting ASOs are demonstrated inExamples 3, 4, and 10; in this case, the compounds are used as AMOs.Examples of degrading ASOs are demonstrated in Example 5 and 12; inthese cases, the compounds are used as anti-mRNA oligonucleotides in anRNase H active design.

In a further embodiment of the invention, the modifications could alsobe incorporated into double-stranded nucleic acids, such as syntheticsiRNAs and miRNAs (miR-mimics). Careful placement of the modifying groupshould lead to improvements in nuclease stability and could alter localthermal stability, which if employed asymmetrically in an RNA duplex, iswell known to influence strand loading into RISC (Peek and Behlke, 2007,Curr. Opin. Mol. Ther. 9(2): 110-18), and therefore impact relativebiological potency of the compound as a synthetic trigger of RNAi.Utility in RNAi applications is demonstrated in Example 6.

Oligonucleotides antisense in orientation to miRNAs will bind the miRNAand functionally remove that species from participation in themicroRNA-Induced Silencing Complex (miRISC) (Krutzfeldt et al., 2007,Nucleic Acids Res. 35(9): 2885-92). Such AMOs are thought to function bya steric binding mechanism, and compounds with high stability and highaffinity generally show improved functional performance compared withlow affinity compounds (Lennox and Behlke, 2010, Pharm. Res. 27(9):1788-99). The ASOs of the present invention can function as anti-miRNAoligonucleotides. This function is demonstrated in Examples 3, 4, 10 and11.

In the modifications of the present invention, a modifying group isinserted terminally or between adjacent bases, thereby generating an ASOwith reduced toxicity and improved binding affinity and nucleasestability. The bases can be DNA, 2′OMe RNA, LNA, or other modifiedbases. However, modified bases do not need to be employed. Low toxicityfor one of the modifying groups of the present invention, thenapthyl-azo modifier, is demonstrated in Example 7. Because themodifications are inserted between the bases, they can be added as aphosphoramidite compound using standard phosphoramidite synthesischemistry. The modifying group can also be placed at the 5′-end, at the3′-end, or at both the 5′-end and 3′-end of the ASO. Modification at the3′-end can be introduced as a modified CPG support or made using aphosphoramidite with a universal support. It is possible to have anon-base modification at both terminal ends, but in many embodimentscontaining more than one non-base modifier it may be preferential toplace one non-base modifier at the terminal end and the second non-basemodifier internally, such as between the terminal and penultimate bases.This simplifies the manufacture of oligonucleotides by allowing forfurther attachment and the base end.

In yet another application where ASOs are employed to alter or modifygene expression, the ASOs are designed to be complementary to a pre-mRNAspecies at sites at or near an intron/exon splice junction. Binding ofthe ASO at or near splice sites can alter processing at this intron/exonjunction by the nuclear splicing machinery thereby changing splicepatterns present in the final mature mRNA (i.e., can be used to alterthe exons that are included or excluded in the final processed mRNA).Following mRNA maturation, the altered mRNA will direct synthesis of analtered protein species as a result of this ASO treatment. Methods todesign splice-blocking oligonucleotides (SBOs) are well known to thosewith skill in the art (see, e.g., Aartsma-Rus et al., 2009, Mol. Ther.17(3): 548-53; and Mitrpant et al., 2009, Mol. Ther. 17(8): 1418-26).Because SBOs are intended to alter the form of an mRNA but not destroythat mRNA, oligonucleotides of this class are made using chemistrieswhich are compatible with steric blocking antisense mechanism of actionand not with chemistries or designs that trigger RNA degradation. Oneexample of the use of SBOs induces exon-skipping in the dystrophin genein individuals having a mutant form of this gene which causes Duchennemuscular dystrophy (see Muntoni and Wood, 2011, Nat. Rev. Drug Discov.10(8): 621-37; and Goemans et al., 2011, N. Engl. J. Med. 364(16):1513-22). Synthetic oligonucleotides using the design and chemistries ofthe present invention can be employed as SBOs. This class of ASO hasalso been called “splice switching oligonucleotides”, or SSOs.

In one embodiment, a synthetic oligonucleotide comprises anon-nucleotide modifier of the present invention positioned at or nearone or both ends of the sequence. In another embodiment, a syntheticoligonucleotide comprises a non-nucleotide modifier of the presentinvention positioned at the 3′ or 5′-end. In a further embodiment, theoligonucleotide contains a first modification at a terminal end and asecond modification between the terminal base and the penultimate baseof the other end. In yet a further embodiment, the oligonucleotidecontains a modification between the terminal base and the penultimatebase at the 5′-end, and a second modification at the 3′-end. Therelative increase of binding affinity contributed by the non-basemodifier may vary with sequence context (Example 8) which can influencewhich of the various design options taught herein is most potent.

In one embodiment of the invention, the modification is a napthyl-azocompound. The oligonucleotide is made using modified bases such that thecomplex of the SBO with the target pre-mRNA does not form a substratefor RNase H, using chemically-modified residues that are well known tothose with skill in the art, including, for example, 2′-O-methyl RNA,2′-methyoxyethyl RNA (2′-MOE), 2′-F RNA, LNA, and the like. SBOs madeusing the non-nucleotide modifiers of the present invention haveincreased binding affinity compared to the cognate unmodified species.This can permit use of shorter sequences, which can show improved uptakeinto cells and improved biological activity.

In another embodiment of the invention, the modification has thestructure:

In a further embodiment of the invention, the modification has thestructure:

The antisense oligonucleotides of the invention may be conjugated toother ligands, which may aid in the delivery of the antisenseoligonucleotide to a cell or organism. In one embodiment of theinvention, the ligand is 5′ cholesterol monoethyleneglycol (/5CholMEG/):

In another embodiment of the invention, the ligand is 5′ cholesteroltriethyleneglycol (/5Chol-TEG/):

In a further embodiment of the invention, the ligand is 3′ cholesterolmonoethyleneglycol (/3CholMEG/):

In another embodiment of the invention, the ligand is 3′ cholesteroltriethyleneglycol (/3CholTEG/):

The ligand may be conjugated to the antisense oligonucleotide with orwithout an additional S18 (hexaethyleneglycol) spacer. In a preferredembodiment, the antisense oligonucleotide is an AMO. In anotherpreferred embodiment, the non-nucleotide modification is a FQnapthyl-azo compound (also referred to as iFQ or ZEN in thisdisclosure).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the improved thermal stability of internalnapthyl-azo-containing oligomers compared to other compounds.

Oligonucleotide Synthesis and Preparation.

DNA oligonucleotides were synthesized using solid phase phosphoramiditechemistry, deprotected and desalted on NAP-5 columns (Amersham PharmaciaBiotech, Piscataway, N.J.) according to routine techniques (Caruthers etal., 1992, Methods Enzymol. 211: 3-20). The oligomers were purifiedusing reversed-phase high performance liquid chromatography (RP-HPLC).The purity of each oligomer was determined by capillary electrophoresis(CE) carried out on a Beckman P/ACE MDQ system (Beckman Coulter, Inc.,Fullerton, Calif.). All single-strand oligomers were at least 90% pure.Electrospray-ionization liquid chromatography mass spectrometry(ESI-LCMS) of the oligonucleotides was conducted using an Oligo HTCSsystem (Novatia, Princeton, N.J.), which consisted of ThermoFinniganTSQ7000, Xcalibur data system, ProMass data processing software, andParadigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.). Protocolsrecommended by the manufacturers were followed. Experimental molarmasses for all single-strand oligomers were within 1.5 g/mol of expectedmolar mass. These results confirm identity of the oligomers.

Preparation of DNA Samples.

Melting experiments were carried out in buffer containing 3.87 mMNaH₂PO₄, 6.13 mM Na₂HPO₄, 1 mM Na₂EDTA, and 1 M NaCl. 1 M NaOH was usedto titrate each solution to pH 7.0. Total sodium concentrations wereestimated to be 1.02 M. The DNA samples were thoroughly dialyzed againstmelting buffer in a 28-well Microdialysis System (Life Technologies,Carlsbad, Calif.) following the manufacturer's recommended protocol.Concentrations of DNA oligomers were estimated from the samples' UVabsorbance at 260 nm in a spectrophotometer (Beckman Coulter, Inc.,Fullerton, Calif.), using extinction coefficients for eacholigonucleotide that were estimated using the nearest neighbor model forcalculating extinction coefficients (see Warshaw et al., 1966, J. Mol.Biol. 20(1): 29-38).

Internal Modifications Studied.

The FQ napthyl-azo compound (Formula 3, Integrated DNA Technologies,Inc., sometimes referred to as “iFQ” or “ZEN” in this disclosure), wasintroduced into oligonucleotides using phosphoramidite reagents at thetime of synthesis.

In the first series of duplexes, the iFQ group was placed as aninsertion between bases in the duplex so that a 10-base top strandannealed to a 10-base bottom strand and the iFQ group was not aligned toa base. Additionally, 10-mer oligonucleotides with C3 spacer insertionswere also synthesized and studied. The C3 spacer represents the controlwherein a linear insertion of a phosphate group plus propanediol isplaced between bases, which is similar to the iFQ insertions withouthaving the napthyl-azo ring structures present. Extinction coefficientsat 260 nm of iFQ were estimated to be 13340; the C3 spacer does notcontribute to UV absorbance.

In a second series of duplexes, the iFQ group was placed as asubstitution for a base in the duplex so that a 9-base top strandannealed to a 10-base bottom strand and the iFQ group was aligned to abase. Additionally, 10-mer oligonucleotides with C3 spacer substitutionswere also synthesized and studied.

Measurement of Melting Curves.

Oligomer concentrations were measured at least twice for each sample. Ifthe estimated concentrations for any sample differed more than 4%, theresults were discarded and new absorbance measurements were performed.To prepare oligonucleotide duplexes, complementary DNA oligomers weremixed in 1:1 molar ratio, heated to 367 K (i.e., 94° C.) and slowlycooled to an ambient temperature. Each solution of duplex DNA wasdiluted with melting buffer to a total DNA concentration (C_(T)) of 2μM.

Melting experiments were conducted on a single beam Beckman DU 650spectrophotometer (Beckman-Coulter) with a Micro T_(m) Analysisaccessory, a Beckman High Performance Peltier Controller (to regulatethe temperature), and 1 cm path-length cuvettes. Melt data were recordedusing a PC interfaced to the spectrophotometer. UV-absorbance values at268 nm wavelength were measured at 0.1 degree increments in thetemperature range from 383 to 368 K (i.e., 10-95° C.). Both heating(i.e., “denaturation”) and cooling (i.e., “renaturation”) transitioncurves were recorded in each sample at a controlled rate of temperaturechange (24.9±0.3° C. per hour). Sample temperatures were collected fromthe internal probe located inside the Peltier holder, and recorded witheach sample's UV-absorbance data. Melting profiles were also recordedfor samples of buffer alone (no oligonucleotide), and these “blank”profiles were digitally subtracted from melting curves of the DNAsamples. To minimize systematic errors, at least two melting curves werecollected for each sample in different cuvettes and in differentpositions within the Peltier holder.

Determination of Melting Temperatures.

To determine each sample's melting temperature, the melting profileswere analyzed using methods that have been previously described (seeDoktycz et al., 1992, Biopolymers 32(7): 849-64; Owczarzy et al., 1997,Biopolymers 44(3): 217-39; and Owczarzy, 2005, Biophys. Chem. 117(3):207-15.). Briefly, the experimental data for each sample was smoothed,using a digital filter, to obtain a plot of the sample's UV-absorbanceas a function of its temperature. The fraction of single-strandedoligonucleotide molecules, θ, was then calculated from that plot. The“melting temperature” or “T_(m)” of a sample was defined as thetemperature where θ=0.5. Table 1 lists the melting temperatures of theoligonucleotides tested.

TABLE 1 Melting temperatures for nucleic acids containinga single internal modifying group as an insertion SEQ ID NO: SequenceT_(m) ΔT_(m) Avg ΔT_(m) 1 5′ ATCGTTGCTA 43.9 — 2 3′ TAGCAACGAT 3 5′ATC/GTTGCTA iFQ ″/″ 48.0 4.1 +3.7 2 3′ TAG CAACGAT 4 5′ ATCG/TTGCTAiFQ ″/″ 48.6 4.7 2 3′ TAGC AACGAT 5 5′ ATCGT/TGCTA iFQ ″/″ 46.3 2.4 2 3′TAGCA ACGAT 6 5′ A/TCGTTGCTA iFQ ″/″ 51.75 7.9 +7.2 2 3′ T AGCAACGAT 75′ ATCGTTGCT/A iFQ ″/″ 50.25 6.4 2 3′ TAGCAACGA T 8 5′ ATC/GTTGCTAiSpC3 ″/″ 36.3 −7.6 −8.7 2 3′ TAG CAACGAT 9 5′ ATCG/TTGCTA iSpC3 ″/″36.6 −7.3 2 3′ TAGC AACGAT 10 5′ ATCGT/TGCTA iSpC3 ″/″ 32.6 −11.3 2 3′TAGCA ACGAT ″/″ signifies the site of insertion of a modifying groupbetween bases as indicated. iFQ = internal FQ azo quencher (ZEN) iSpC3 =internal C3 spacer

When the iFQ (ZEN) modifier was inserted centrally within a 10-meroligonucleotide (between bases 3/4 , 4/5, or 5/6), T_(m) was increasedby an average of 3.7° C. When placed between terminal residues (betweenbases 1/2 or 9/10), T_(m) was increased by an average of 7.2° C. Incontrast, insertion of a small propanediol group (C3 spacer) had asignificant negative impact on the T_(m) of the duplex (average ΔT_(m)of −8.7° C.).

A subset of these sequences were studied using the internalmodifications as base substitutions, such that now a 9-base top strandannealed to a 10-base bottom strand with the modification replacing abase and being aligned with a base on the opposing strand. Results areshown in Table 2. In this case, it is evident that the base substitutionwas significantly destabilizing whereas the insertions (Table 1) werestabilizing (ZEN) or were at least less destabilizing (C3).

TABLE 2 Melting temperatures for nucleic acids containinga single internal modifying group comparing substitution vs. insertionIns SEQ ID vs. NO: Duplex Sequence Subs T_(m) ΔT_(m) 1 5′-ATCGTTGCTA-3′— 43.9 0.0 2 3′-TAGCAACGAT-5′ 3 5′-ATC/GTTGCTA-3′ iFQ ″/″ I 48.0 4.1 23′-TAG CAACGAT-5′ 8 5′-ATC/GTTGCTA-3′ iSpC3 ″/″ I 36.3 −7.6 23′-TAG CAACGAT-5′ 11 5′-ATC/TTGCTA-3′ iFQ ″/″ S 34.7 −9.2 23′-TAGCAACGAT-5′ 12 5′-ATC/TTGCTA-3′ iSpC3 ″/″ S <20 2 3′-TAGCAACGAT-5′13 5′-ATCG/TTGCTA-3′ iFQ ″/″ I 48.6 4.7 2 3′-TAGC AACGAT-5′ 145′-ATCG/TTGCTA-3′ iSpC3 ″/″ I 36.6 −7.3 2 3′-TAGC AACGAT-5′ 155′-ATCG/TGCTA-3′ iFQ ″/″ S 38.2 −5.7 2 3′-TAGCAACGAT-5′ 165′-ATCG/TGCTA-3′ iSpC3 ″/″ S <24 2 3′-TAGCAACGAT-5′ 17 5′-ATCGT/TGCTA-3′iFQ ″/″ I 46.3 2.4 2 3′-TAGCA ACGAT-5′ 18 5′-ATCGT/TGCTA-3′ iSpC3 ″/″ I32.6 −11.3 2 3′-TAGCA ACGAT-5′ 19 5′-ATCGT/GCTA-3′ iFQ ″/″ S 40.8 −3.1 23′-TAGCAACGAT-5′ 20 5′-ATCGT/GCTA-3′ iSpC3 ″/″ S <26 2 3′-TAGCAACGAT-5′

For this series of internal modifications, the average ΔT_(m) for iFQ(ZEN) insertion was +3.7° C. while the average ΔT_(m) for iFQ (ZEN)substitution was −6° C. The average ΔT_(m) for iC3 spacer insertion was−8.7° C. while the average ΔT_(m) for iC3 spacer substitution was morethan −20° C. (accurate measurements were not possible as the T_(m) wasbelow room temperature). Therefore insertion placement is preferred tosubstitution placement.

The napthyl-azo modifier was introduced into the same 10-mer oligomersequence at 2 or 3 sites, either adjacent to or separated by severalbases. Duplexes were formed and T_(m) values were measured as before.Results are shown in Table 3. Some of the singly modified duplexes fromTable 1 are also included in Table 3 to improve clarity of comparisonsbetween modification patterns.

TABLE 3 Melting temperatures for nucleic acids containingmultiple internal modifying groups as insertions SEQ ID NO: Sequence TmΔTm 1 5′ ATCGTTGCTA 43.87 — 2 3′ TAGCAACGAT 3 5′ ATC/GTTGCTA 1x iFQ ″/″48.02 4.15 2 3′ TAG CAACGAT 21 5′ ATC//GTTGCTA 2x iFQ ″//″ 39.62 −4.25 23′ TAG CAACGAT 22 5′ ATC/GTT/GCTA 2x iFQ ″/.../″ 46.72 2.85 2 3′TAG CAA CGAT 23 5′ ATC/GT/TG/CTA 3x iFQ ″/../../″ 43.36 −0.51 2 3′TAG CA AC GAT 13 5′ ATCG/TTGCTA 1x iFQ ″/″ 48.57 4.70 2 3′ TAGC AACGAT24 5′ ATCG//TTGCTA 2x iFQ ″//″ 39.82 −4.05 2 3′ TAGC  AACGAT 17 5′ATCGT/TGCTA 1x iFQ ″/″ 46.32 2.45 2 3′ TAGCA ACGAT 25 5′ ATCGT//TGCTA2x iFQ ″/″ 36.76 −8.90 2 3′ TAGCA  ACGAT 26 5′ A/TCGTTGCTA 1x iFQ ″/″51.75 7.88 2 3′ T AGCAACGAT 27 5′ ATCGTTGCT/A 1x iFQ ″/″ 50.25 6.38 2 3′TAGCAACGA T 28 5′ A/TCGTTGCT/A 2x iFQ ″/″ 54.91 11.04 2 3′ T AGCAACGA T″/″ signifies the site of insertion of a modifying group between basesas indicated.

Insertion of two adjacent napthyl-azo modifiers was destabilizing andT_(m) was found to change by −4 to −8.9° C. depending on sequencecontext. Placing two napthyl-azo modifying groups in the same sequenceseparated by 3 bases was slightly stabilizing (T_(m)+2.9° C.); however,this was less stabilizing than use of a single modifier alone(T_(m)+4.7° C.). Use of 3 modifier groups separated by 2 bases betweengroups was destabilizing. However, when two napthyl-azo modifier groupswere placed at the ends (between both bases 1/2 and 9/10), T_(m) wasincreased by 11° C. Thus, an additive effect can be obtained by placingmultiple insertions of the modifying group into a sequence so long as asufficient number of bases separate the groups. End effects areparticularly potent.

Therefore, internal incorporation of the napthyl-azo group within a DNAduplex stabilizes the duplex when placed as an insertion between bases.Certain anthraquinone groups can stabilize a duplex when placed on theends (Patra et al., 2009, J. Am. Chem. Soc. 131(35): 12671-81); however,this effect has not been described for internal placement. Therefore,the use of napthyl-azo-class compounds would be preferred as an internalmodifying group to increase duplex stability.

EXAMPLE 2

This example demonstrates the improved nuclease stability of internalnapthyl-azo-containing oligomers compared to other compounds.

Oligonucleotide Synthesis and Purification.

DNA oligonucleotides were synthesized using solid phase phosphoramiditechemistry, deprotected and desalted on NAP-5 columns (Amersham PharmaciaBiotech, Piscataway, N.J.) according to routine techniques (Caruthers etal., 1992). The oligomers were purified using reversed-phase highperformance liquid chromatography (RP-HPLC). The purity of each oligomerwas determined by capillary electrophoresis (CE) carried out on aBeckman P/ACE MDQ system (Beckman Coulter, Inc., Fullerton, Calif.). Allsingle-strand oligomers were at least 90% pure. Electrospray-ionizationliquid chromatography mass spectrometry (ESI-LCMS) of theoligonucleotides was conducted using an Oligo HTCS system (Novatia,Princeton, N.J.), which consisted of ThermoFinnigan TSQ7000, Xcaliburdata system, ProMass data processing software, and Paradigm MS4™ HPLC(Michrom BioResources, Auburn, Calif.). Protocols recommended by themanufacturers were followed. Experimental molar masses for allsingle-strand oligomers were within 1.5 g/mol of expected molar mass.These results confirm identity of the oligomers. The synthesizedoligonucleotides are listed in Table 4.

TABLE 4 Synthetic oligomers employed in Example 2 SEQ ID NO: NameSequence 1 DNA 5′ ATCGTTGCTA 3′ 26 5′ iFQ 5′ A(iFQ)TCGTTGCTA 3′ 27 3′iFQ 5′ ATCGTTGCT(iFQ)A 3′ 28 5′ + 3′ iFQ 5′ A(iFQ)TCGTTGCT(iFQ)A 3′ 295′ iC3 5′ A(iSpC3)TCGTTGCTA 3′ 30 3′ iC3 5′ ATCGTTGCT(iSpC3) A 3′ 315′ + 3′ iC3 5′ A(iSpC3)TCGTTGCT(iSpC3) A 3′

Radiolabeling of Oligomers.

Single-stranded oligomers were radiolabeled at the 5′-end usingpolynucleotide kinase. Briefly, 5 pmoles of each oligonucleotide wereincubated with 10 units of OptiKinase (USB, Cleveland, Ohio) and 10pmoles of alpha ³²P γ-ΔTP (3000 Ci/mmol) (Perkin Elmer, Waltham, Mass.)for 30 minutes at 37° C., followed by 65° C. for 10 minutes. Excessradionucleotide was removed by gel filtration using two sequentialpasses through MicroSpin G-25 columns (GE Healthcare, Buckinghamshire,UK). Isotope incorporation was measured in a Perkin Elmer TriCarb 2800TR scintillation counter (Perkin Elmer, Waltham, Mass.).

Serum Degradation of Oligomers.

As labeling efficiencies varied (lower specific activity was obtainedfor the oligomers with a modification near the 5′-end), equivalentnumbers of dpms of radiolabeled oligomers were mixed with unlabeledoligomers to a final concentration of 8 μM in the presence of 50% fetalbovine serum (not heat inactivated; Invitrogen, Carlsbad, Calif.).Samples were incubated at 37° C. for 0, 30, 60, or 240 minutes; aliquotswere removed at the indicated time points, an equal volume of 90%formamide was added, and samples flash frozen on dry ice. Degradationproducts were separated by PAGE using a 20% polyacrylamide, 7 M Ureadenaturing gel and visualized on a Cyclone phosphorimager (Perkin Elmer,Waltham, Mass.). Results are shown in FIG. 1.

The unmodified DNA oligomer was rapidly degraded and no intactfull-length material was present after 30 minutes incubation. The samplewas fully degraded by 4 hours. A similar pattern of degradation was seenfor the oligomer having a single internal C3 spacer positioned near the5′-end. In contrast, only incomplete degradation was observed for theoligomer bearing a single internal FQ modifier near the 5′-end. Thedegradation pattern observed is most consistent with processive3′-exonuclease cleavage that stopped before the oligomer was fullydegraded. This suggests the possibility that the iFQ modifier protectsneighboring DNA residues from exonuclease degradation, providing a smallzone of protection around the 5′-end.

The oligomer having a single internal C3 spacer near the 3′-end showsprompt removal of what appears to be a single base and then was slowlydegraded. Slightly greater protection was seen in the oligomer having aninternal C3 spacer placed near both ends. In contrast, no evidence wasseen for single base cleavage at the 3′-end of the oligomer having asingle internal FQ modifier near the 3′-end, and no evidence fordegradation was observed after 4 hours incubation in 50% serum for theoligomer having an internal FQ modifier placed near both ends.

Therefore, the FQ modifier will block exonuclease attack from theenzymes present in fetal bovine serum, and can confer relative nucleaseresistance to neighboring unmodified bases, creating a protected “zone”in its vicinity.

EXAMPLE 3

This example demonstrates improved functional activity of internalnapthyl-azo-containing ASOs at reducing microRNA activity compared toother compounds.

Oligonucleotide Synthesis and Purification.

DNA, 2′OMe RNA, and LNA containing oligonucleotides were synthesizedusing solid phase phosphoramidite chemistry, deprotected and desalted onNAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.) accordingto routine techniques (Caruthers et al., 1992). The oligomers werepurified using reversed-phase high performance liquid chromatography(RP-HPLC). The purity of each oligomer was determined by capillaryelectrophoresis (CE) carried out on a Beckman P/ACE MDQ system (BeckmanCoulter, Inc., Fullerton, Calif.). All single-strand oligomers were atleast 85% pure. Electrospray-ionization liquid chromatography massspectrometry (ESI-LCMS) of the oligonucleotides was conducted using anOligo HTCS system (Novatia, Princeton, N.J.), which consisted ofThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processingsoftware, and Paradigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.).Protocols recommended by the manufacturers were followed. Experimentalmolar masses for all single-strand oligomers were within 1.5 g/mol ofexpected molar mass. These results confirm identity of the oligomers.Table 5 lists the synthetic oligomers used in this Example.

TABLE 5 Synthetic oligomers employed in Example 3 (miR-21 AMOs) SEQ IDNO: Name Sequence 32 2′OMe U C A A C A U C A G U C U G A U A A G C U A33 2′OMe PSends U*C*A*A C A U C A G U C U G A U A A G*C*U*A 34 2′OMe PSU*C*A*A*C*A*U*C*A*G*U*C*U*G*A*U*A*A*G*C*U*A 35 2′OMe 5′ + 3′iFQ

C A A C A U C A G U C U G A U A A G C 

A 36 2′OMePS 5′ + 3′iFQ

C*A*A*C*A*U*C*A*G*U*C*U*G*A*U*A*A*G*C*

A 37 2′OMe 3′iFQ U C A A C A U C A G U C U G A U A A G C 

A 38 2′OMe 5′iFQ

C A A C A U C A G U C U G A U A A G C U A 39 2′Me 5′ + I + 3′iFQ

C A A C A U C A G 

C U G A U A A G C 

A 40 DNA/LNA PS t*C*a*a*C*a*t*C*a*g*T*c*t*G*a*t*A*a*g*C*t*a 412′OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C*U*G*A*U*A*A*G*C*U*A Uppercase =2′OMe RNA Lowercase = DNA Uppercase with underscore = LNA ″*″ =phosphorothioate linkage ″

″ = napthyl-azo modifier (iFQ)

Plasmid Preparation.

The psiCHECK™-2 vector (Promega, Madison, Wis.) was restriction enzymedigested sequentially with XhoI and NotI (New England Biolabs, Ipswitch,Mass.) and purified with a Qiaquick PCR purification column (Qiagen,Valencia, Calif.). A perfect complement hsa-miR-21 binding site wascreated by annealing two synthetic duplexed oligonucleotides (IntegratedDNA Technologies, Coralville, Iowa) and was cloned into the XhoI/NotIsites in the 3′UTR of Renilla luciferase. This miR-21 reporter constructwas sequence verified on a 3130 Genetic Analyzer (AB, Foster City,Calif.). Plasmids were purified using a Plasmid Midiprep Kit (Bio-Rad,Hercules, Calif.) and treated twice for endotoxin removal with theMiraCLEAN Endotoxin Removal Kit (Mirus Corporation, Madison, Wis.).Plasmids were filtered through a 0.2μ filter and quantified bymeasurement of the absorbance at 260 nm using UV spectrophotometry. Thisreporter plasmid having a perfect match miRNA binding site is denoted aspsiCHECK™-2-miR21.

Cell Culture, Transfections, and Luciferase Assays.

HeLa cells were plated in a 100 mm dish in DMEM containing 10% FBS toachieve 90% confluency the next day. The following morning, 5 μg of thepsiCHECK™-2-miR21 plasmid was transfected with Lipofectamine™ 2000(Invitrogen, Carlsbad, Calif.). After 6 hours, cells were washed with1×PBS, trypsinized, counted, and replated in DMEM with 10% FBS in48-well plates to achieve ˜70% confluency the next day. The followingmorning, the miR-21 AMOs were transfected at various concentrations intriplicate with 1 μl TriFECTin® (Integrated DNA Technologies) per wellin DMEM without serum. After 6 hours, the transfection media was removedand replenished with DMEM containing 10% FBS. The following morning, (48hours after plasmid transfection, 24 hours after miRNA AMO transfection)the cells were analyzed for luciferase luminescence using theDual-Luciferase® Reporter Assay System (Promega, Madison, Wis.) per themanufacturer's instructions. Renilla luciferase was measured as a foldincrease in expression compared to the TriFECTin® reagent-only negativecontrols. Values for Renilla luciferase luminescence were normalized tolevels concurrently measured for firefly luciferase, which is present asa separate expression unit on the same plasmids as an internal control(RLuc/FLuc ratio).

Results.

The RLuc/FLuc ratios obtained from transfections done with the variousAMOs are shown in FIG. 2. In the untreated state, HeLa cells containlarge amounts of miRNA 21 that suppress expression of the RLuc reporter.Any treatment that decreases miR-21 levels leads to an increase in RLucexpression and thus increases the relative RLuc/FLuc ratio (with FLucserving as an internal normalization control for transfectionefficiency).

The unmodified 2′OMe RNA AMO showed essentially no inhibition of miR-21activity, probably due to rapid nuclease degradation of this unprotectedoligomer during transfection or in the intracellular environment. Theaddition of 3 PS linkages on each end of the AMO blocks exonucleaseattack and the “2′OMe-PSends” AMO showed good potency for functionalknockdown of miR-21. When this AMO is changed to be fully PS modified(“2′OMe-PS”), potency drops, which is probably due to having lowerbinding affinity (lower T_(m)) that accompanies extensive PSmodification. Each substitution of a PS bond for a standardphosphodiester bond reduces T_(m), and there are 21 PS bonds in thisoligomer compared with only 6 PS bonds in the “2′OMe PSends” version.

A desirable modification chemistry or modification pattern is one thatboth increases nuclease stability and increases T_(m). The internalnapthyl-azo modifier meets these criteria. The 2′OMe oligomer having aninternal napthyl-azo modifier placed between the terminal and adjacentbases on each end (2′OMe 5′+3′ iFQ) showed markedly improved anti-miR21activity and was more potent than any of the PS modified 2′OMe AMOstested. Adding PS modification to this design (2′OMePS 5′+3′ iFQ)reduced potency, likely due to the lower binding affinity caused by theaddition of 19 PS linkages. This compound was nevertheless stillsignificantly more potent than the 2′OMe-PS version without the 2 iFQmodifications.

Protecting only one end of the anti-miR-21 AMO with an internalnapthyl-azo modifier showed improved potency when compared with theunmodified 2′OMe AMO; however, the performance was much reduced comparedwith the dual-modified version. Interestingly, modification at the5′-terminal linkage had more effect than modification at the 3′-terminallinkage, the exact opposite of the results anticipated from the relativeserum stability profiles demonstrated in Example 2. This result isexplained by measured effects of T_(m) (see Table 6).

Addition of a third iFQ modification into the end-blocked version (2′OMe5′+I+3′ iFQ) showed reduced potency compared with the originalend-blocked version (2′OMe 5′+3′ iFQ), which is likely due to areduction of T_(m) seen with placing this many iFQ modifying groups in asingle, short 22-mer sequence.

The “DNA/LNA-PS” AMO is a design employed by Exiqon as its preferredanti-miRNA agent and is widely accepted as the “gold standard” for miRNAknockdown studies performed today. The DNA/LNA compound showed the samepotency as the dual-modified “2′OMe 5′+3′ iFQ” AMO. The “2′OMe/LNA-PS”AMO showed highest potency within the set studied. The LNA modificationconfers nuclease resistance and gives very large increases in T_(m),resulting in AMOs with higher potency but also having lower specificitythan AMOs without LNA bases with lower binding affinity. The relativespecificity of the different AMOs is presented in Example 4 below. Ofnote, the LNA-PS modified AMOs show some toxicity and cell culturestransfected with the highest doses (50 nM) had dysmorphic, unhealthyappearing cells at the time of harvest. The “2′OMe 5′+3′ iFQ” AMO didnot show any visual evidence for toxicity at any of the doses tested. Insubsequent experimentation, toxicity effects were evaluated at highdoses by measuring cell viability, cytotoxicity, and induction ofapoptosis (see Example 7). The “2′OMe 5′+3′ iFQ” chemistry showed nocellular toxicity, compared to the substantial cellular toxicity thatoccurred upon transfection of single-stranded oligonucleotidescontaining LNA bases, extensive PS modification (all 21 linkages), orboth LNA and PS modifications (the “gold standard” AMO). Thus, the“2′OMe 5′+3′ iFQ” may be a new class of AMO that achieves high potencyyet maintains low toxicity.

The melting temperatures, T_(m), of the AMOs described above weremeasured using the same methods described in Example 1. Synthetic AMOoligonucleotides were annealed to a synthetic RNA complement (maturemiR21 sequence). Measurements were done at 2 μM duplex concentration in150 mM NaCl to approximate intracellular ion concentration.

TABLE 6 T_(m) of synthetic miR-21 AMOs in 150 mM NaCl SEQ ID NO: NameSequence T_(m) ΔT_(m) 32 2′OMeU C A A C A U C A G U C U G A U A A G C U A 72.1 — 33 2′OMeU*C*A*A C A U C A G U C U G A U A A G*C*U*A 70.9 −1.2 PSends 34 2′OMe PSU*C*A*A*C*A*U*C*A*G*U*C*U*G*A*U*A*A*G*C*U*A 67.1 −5.0 35 2′OMe

C A A C A U C A G U C U G A U A A G C 

A 75.4 +3.3 5′ + 3′iFQ 36 2′OMePS

C*A*A*C*A*U*C*A*G*U*C*U*G*A*U*A*A*G*C*

A 70.6 −1.5 5′ + 3′iFQ 37 2′OMe 3′iFQU C A A C A U C A G U C U G A U A A G C 

A 72.4 +0.3 38 2′OMe 5′iFQ

C A A C A U C A G U C U G A U A A G C U A 74.3 +2.2 39 2′OMe

C A A C A U C A G 

C U G A U A A G C 

A 71.3 −0.8 5′ + I + 3′iFQ 40 DNA/LNAt*C*a*a*C*a*t*C*a*g*T*c*t*G*a*t*A*a*g*C*t*a 74.0 +1.9 PS 41 2′OMe/LNAU*C*A*A*C*A*U*C*A*G*T*C*U*G*A*U*A*A*G*C*U*A 85.9 +13.8 PS Uppercase =2′OMe RNA Lowercase = DNA Uppercase with underscore = LNA ″*″ =phosphorothioate linkage ″

″ = insertion of napthyl-azo modifier (iFQ)

The 22-mer 2′OMe miR21 AMO showed a T_(m) of 72.1° C. when hybridized toan RNA perfect complement in 150 mM NaCl. Substitution of 6 PS bonds fornative PO linkages lowered T_(m) by 1.2° C. (“2′OMe PSends”) andcomplete PS modified lowered T_(m) by 5.0° C. (“2′OMe PS”), a change of−0.20 to −0.25° C. per modified internucleotide linkage. In contrast,insertion of an iFQ group at the 3′-terminal linkage (“2′OMe 3′ iFQ”)resulted in a T_(m) increase of +0.3° C. and at the 5′-terminal linkage(“2′OMe 5′ iFQ”) resulted in a T_(m) increase of +2.2° C. Combiningthese two designs, addition of two iFQ modifications (one at eachterminal linkage, “2′OMe 5′+3′ iFQ”) increased T_(m) to 75.4° C., whichis a change of +3.3° C. compared with the unmodified sequence or +4.5°C. relative to the PS-end blocked sequence (which is the most relevantcomparison). This dual-end-modification pattern results in good nucleaseresistance (FIG. 1) and when employed in a 2′OMe AMO shows increasedT_(m) (Table 6) and is a very potent anti-miR21 agent (FIG. 2).Interestingly, addition of a third iFQ group centrally placed (“2′OMe5′+I+3′ iFQ”) resulted in a T_(m) decrease of 0.8° C. relative to theunmodified compound, or a decrease of 4.1° C. relative to the two-endinsertion version (“2′OMe 5′+3′ iFQ”). Thus while inserting the iFQmodifier between terminal bases increases T_(m), adding a thirdmodification in the center of the sequence leads to a decrease in T_(m),even though these modifications are fully 10 bases distant from eachother. This loss of T_(m) results in a loss of functional potency (FIG.2). Therefore the dual-modified end-insertion pattern is preferred.

As a general rule, the relative potency of the various miR21 AMOscorrelated with increased binding affinity (T_(m)). All variations inpotency observed between compounds could be explained by relativecontributions of improvements in binding affinity and nuclease stabilitybetween the different modification patterns studied. The AMO having2′OMe bases with an iFQ modification placed near each end (“2′OMe 5′+3′iFQ”) provided an excellent balance of nuclease stability with increasedT_(m) and the only AMO showing higher potency was the “2′OMe/LNA-PS”compound. The “2′OMe/LNA-PS” compound, however, showed reducedspecificity due to its extreme elevation in binding affinity (seeExample 4) and increased cellular toxicity (see Example 7). Therefore,the novel “2′OMe 5′+3′ iFQ” design of the present invention is superior.

EXAMPLE 4

This example demonstrates improved specificity of internalnapthyl-azo-containing oligomers when reducing microRNA activitycompared to other compounds containing modifications that increasebinding affinity.

Three of the more potent AMO designs from the functional study performedin Example 3 were examined in greater detail to assess their relativeability to discriminate mismatches between the synthetic anti-miRNAoligonucleotide and their target. In general, high affinityoligonucleotides show high potency but usually show reduced specificityas the high affinity permits hybridization even in the presence of oneor more mismatches in complementarity. The designs “2′OMe 5′+3′ iFQ”,“DNA/LNA-PS”, and “2′OMe/LNA-PS” were synthesized as variants having 1,2, or 3 mismatches to the miR-21 target sequence. Sequences are shown inTable 7. Studies were performed as described in Example 3.

TABLE 7 SEQ ID NO: Name Sequence 35 2′OMe 5′ + 3′iFQ

C A A C A U C A G U C U G A U A A G C 

A 42 2′OMe 5′ + 3′iFQ

C A A C A U C A G U C U 

 A U A A G C 

A 1MUT 43 2′OMe 5′ + 3′iFQ

C A A 

 A U C A G U C U 

 A U A A G C 

A 2MUT 44 2′OMe 5′ + 3′iFQ

C A A 

 A U C A G U C U 

 A U A A G 

 

A 3MUT 40 DNA/LNA PS t*C*a*a*C*a*t*C*a*g*T*c*t*G*a*t*A*a*g*C*t*a 45DNA/LNA PS t*C*a*a*C*a*t*C*a*g*T*c*t*

*a*t*A*a*g*C*t*a 1MUT 46 DNA/LNA PS t*C*a*a*

*a*t*C*a*g*T*c*t*

*a*t*A*a*g*C*t*a 2MUT 47 DNA/LNA PS t*C*a*a*

*a*t*C*a*g*T*c*t*

*a*t*A*a*g*

*t*a 3MUT 41 2′OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C*U*G*A*U*A*A*G*C*U*A 482′OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C*U*

*A*U*A*A*G*C*U*A 1MUT 49 2′OMe/LNA PS U*C*A*A*

*A*U*C*A*G*T*C*U*

*A*U*A*A*G*C*U*A 2MUT 50 2′OMe/LNA PS U*C*A*A*

*A*U*C*A*G*T*C*U*

*A*U*A*A*G*

*U*A 3MUT Uppercase = 2′OMe RNA Lowercase = DNA Uppercase withunderscore = LNA ″*″ = phosphorothioate linkage ″

″ = napthyl-azo modifier (iFQ) Mutations are identified with bold italicfont

Results.

The RLuc/FLuc ratios obtained from transfections done with the variousAMOs are shown in FIG. 3. In the untreated state, HeLa cells containlarge amounts of miRNA 21 that suppress expression of the RLuc reporter.Any treatment that decreases miR-21 levels leads to an increase in RLucexpression and thus increases the relative RLuc/FLuc ratio (with FLucserving as an internal normalization control for transfectionefficiency). For each of the chemistries studied, the parent wild-typesequence is followed by variants having 1, 2, or 3 mutations.

In all cases, the perfect match AMO showed significant suppression ofmiR-21 activity as evidenced by increases in luciferase levels (increasein the RLuc to FLuc ratio indicating de-repression of the RLuc mRNA). Asin Example 3 (FIG. 3), the “2′OMe/LNA-PS” compound showed the highestpotency as evidenced by suppression of miR-21 at low dose (1 nM and 5 nMdata points). The “2′OMe 5′+3′ iFQ” and “DNA/LNA-PS” AMOs showedrelatively similar performance both in wild-type (perfect match) andmutant (mismatch) versions. In both cases, a single mismatch showed apartial reduction of anti-miR-21 activity, the double mismatch showedalmost complete elimination of anti-miR-21 activity, and the triplemismatch did not show any anti-miR-21 activity. In contrast, the higheraffinity “2′OMe/LNA-PS” compound showed significant anti-miR-21 activityfor both the single and double mismatch compounds and even showed someactivity at high dose (50 nM) for the triple mismatch compound. Thus,while the “2′OMe/LNA-PS” compound is most potent, it is also the leastspecific of the reagents studied.

Of note, the above experiments were performed using AMOs that placed themismatches at positions that are LNA modified (in the LNA containingAMOs). This design may influence the likelihood that a mismatch willaffect activity as it disrupts a high affinity LNA:RNA base pair. Thus,these results represent the best case scenario for specificity of theLNA-modified AMOs. The experiment was repeated using a new set ofreagents where the mismatches were all positioned at non-LNA bases. Thisnew series of AMO reagents is shown in Table 8.

TABLE 8 Synthetic oligomers employed in Example 4 (miR-21 AMOs) SEQ IDNO: Name Sequence 35 2′OMe 5′ + 3′iFQ

C A A C A U C A G U C U G A U A A G C 

A 51 2′OMe 5' + 3′iFQ

C A A C A U C A G U C 

 G A U A A G C 

A 1MUT v2 52 2′OMe 5′ + 3′iFQ

C A A C 

 U C A G U C 

 G A U A A G C 

A 2MUT v2 53 2′OMe 5′ + 3′iFQ

C A A C 

 U C A G U C 

 G A U A A 

 C 

A 3MUT v2 40 DNA/LNA PS t*C*a*a*C*a*t*C*a*g*T*c*t*G*a*t*A*a*g*C*t*a 54DNA/LNA PS 1MUT t*C*a*a*C*a*t*C*a*g*T*c*

*G*a*t*A*a*g*C*t*a v2 55 DNA/LNA PS 2MUT t*C*a*a*C*

*t*C*a*g*T*c*

*G*a*t*A*a*g*C*t*a v2 56 DNA/LNA PS 3MUT t*C*a*a*C*

*t*C*a*g*T*c*

*G*a*t*A*a*

*C*t*a v2 41 2′OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C*U*G*A*U*A*A*G*C*U*A 572′OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C*

*G*A*U*A*A*G*C*U*A 1MUT v2 58 2′OMe/LNA PS U*C*A*A*C*

*U*C*A*G*T*C*

*G*A*U*A*A*G*C*U*A 2MUT v2 59 2′OMe/LNA PS U*C*A*A*C*

*U*C*A*G*T*C*

*G*A*U*A*A*

*C*U*A 3MUT v2 Uppercase = 2′OMe RNA Lowercase = DNA Uppercase withunderscore = LNA ″*″ = phosphorothioate linkage ″

″ = napthyl-azo modifier (iFQ) Mutations are identified with bold italicfont

Results.

The RLuc/FLuc ratios obtained from transfections done with the variousAMOs are shown in FIG. 4. In the untreated state, HeLa cells containlarge amounts of miRNA 21 that suppress expression of the RLuc reporter.Any treatment that decreases miR-21 levels leads to an increase in RLucexpression and thus increases the relative RLuc/FLuc ratio (with FLucserving as an internal normalization control for transfectionefficiency). For each of the chemistries studied, the parent wild-typesequence is followed by variants having 1, 2, or 3 mutations.

The results were nearly identical to those obtained with the originalmutation mismatch placement (FIG. 3). In all cases, the perfect matchAMO showed significant suppression of miR-21 activity as evidenced byincreases in luciferase levels (increase in the RLuc to FLuc ratioindicating de-repression of the RLuc mRNA). As in Example 3 (FIG. 3),the “2′OMe/LNA-PS” compound showed the highest potency as evidenced bysuppression of miR-21 at low dose (1 nM and 5 nM data points). The“2′OMe 5′+3′ iFQ” and “DNA/LNA-PS” AMOs showed relatively similarperformance both in wild-type (perfect match) and mutant (mismatch)versions. In both cases, a single mismatch showed a partial reduction ofanti-miR-21 activity, the double mismatch showed almost completeelimination of anti-miR-21 activity, and the triple mismatch did notshow any anti-miR-21 activity. In contrast, the higher affinity“2′OMe/LNA-PS” compound showed significant anti-miR-21 activity for boththe single and double mismatch compounds and even showed some activityat high dose (50 nM) for the triple mismatch compound. Thus, while the“2′OMe/LNA-PS” compound is most potent, it is also the least specific ofthe reagents studied.

EXAMPLE 5

This example demonstrates improved functional activity of internalnapthyl-azo-containing oligomers at reducing cellular mRNA levels whenincorporated into RNase H active ASOs as compared to other relatedcompounds.

Oligonucleotides antisense in orientation to cellular messenger RNAs(mRNAs) will hybridize to the mRNA and form an RNA/DNA heteroduplex,which is a substrate for cellular RNase H. Degradation by RNase H leadsto a cut site in the mRNA and subsequently to total degradation of thatRNA species, thereby functionally lowering effective expression of thetargeted transcript and the protein it encodes. ASOs of this typerequire a domain containing at least 4 bases of DNA to be a substratefor RNase H, and maximal activity is not seen until 8-10 DNA bases arepresent. ASOs must be chemically modified to resist degradation by serumand cellular nucleases. Phosphorothioate (PS) modification of theinternucleotide linkages is compatible with RNase H activation, howevermost other nuclease resistant modifications prevent RNase H activity,including all 2′-modifications, such as 2′OMe RNA, LNA, MOE, etc. The PSmodification lowers binding affinity (T_(m)). In general, modificationsthat lower T_(m) decrease potency while modifications that increaseT_(m) improve potency. One strategy to improve potency of ASOs is toemploy a chimeric design where a low T_(m), RNase H activating domainmade of PS-modified DNA is flanked by end domains that contain2′-modified sugars which confer high binding affinity but are not RNaseH activating (“gapmer” design). One commonly employed strategy is toplace five 2′-modified bases at the 5′-end, ten PS-modified DNA bases inthe middle, and five 2′-modified bases at the 3′-end of the ASO (socalled “5-10-5” design). A modification that confers nucleaseresistance, increases binding affinity, and does not impair thereagent's ability to activate RNase H would be ideal. The presentexample demonstrates the utility of the internal napthyl-azo modifier toimprove the nuclease stability and increase binding affinity of ASOs,enhancing their function as gene knockdown reagents.

Oligonucleotide Synthesis and Purification.

DNA, 2′OMe RNA, and LNA containing oligonucleotides were synthesizedusing solid phase phosphoramidite chemistry, deprotected and desalted onNAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.) accordingto routine techniques (Caruthers et al., 1992). The oligomers werepurified using reversed-phase high performance liquid chromatography(RP-HPLC). The purity of each oligomer was determined by capillaryelectrophoresis (CE) carried out on a Beckman P/ACE MDQ system (BeckmanCoulter, Inc., Fullerton, Calif.). All single-strand oligomers were atleast 85% pure. Electrospray-ionization liquid chromatography massspectrometry (ESI-LCMS) of the oligonucleotides was conducted using anOligo HTCS system (Novatia, Princeton, N.J.), which consisted ofThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processingsoftware, and Paradigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.).Protocols recommended by the manufacturers were followed. Experimentalmolar masses for all single-strand oligomers were within 1.5 g/mol ofexpected molar mass. These results confirm identity of the oligomers.

TABLE 9 Synthetic oligomers employed in Example 5 (anti-HPRT ASOs) SEQID NO: Name Sequence 60 HPRT#1 DNAa t a g g a c t c c a g a t g t t t c c 61 HPRT#1 DNA 5′iFQ

t a g g a c t c c a g a t g t t t c c 62 HPRT#1 DNA 3′iFQa t a g g a c t c c a g a t g t t t 

c 63 HPRT#1 DNA 5′ + 3′iFQ

t a g g a c t c c a g a t g t t t 

c 64 HPRT#1 DNA 5′ + i + 3′iFQ

t a g g a c t c 

a g a t g t t t 

c 65 HPRT#1 DNA PS a*t*a*g*g*a*c*t*c*c*a*g*a*t*g*t*t*t*c*c 66HPRT#1 DNA PS 5′iFQ

t*a*g*g*a*c*t*c*c*a*g*a*t*g*t*t*t*c*c 67 HPRT#1 DNA PS 3′iFQa*t*a*g*g*a*c*t*c*c*a*g*a*t*g*t*t*t*

c 68 HPRT#1 DNA PS 5′ + 3′iFQ

t*a*g*g*a*c*t*c*c*a*g*a*t*g*t*t*t*

c 69 HPRT#1 DNA PS

t*a*g*g*a*c*t*c*

a*g*a*t*g*t*t*t*

c 5′ + I + 3′iFQ 70 HPRT#1 5-10-5A U A G G a c t c c a g a t g U U U C C 71 HPRT#1 5-10-5 2x iFQ

U A G G a c t c c a g a t g U U U 

C 72 HPRT#1 5-10-5 3x iFQ

U A G G a c t c 

a g a t g U U U 

C 73 HPRT#1 5-10-5 PS A*U*A*G*G*a*c*t*c*c*a*g*a*t*g*U*U*U*C*C 74HPRT#1 5-10-5 PS 2x iFQ

U*A*G*G*a*c*t*c*c*a*g*a*t*g*U*U*U*

C 75 HPRT#1 5-10-5 gapPS A U A G G a*c*t*c*c*a*g*a*t*g*U U U C C 76HPRT#1 5-10-5 gapPS 2x

U A G G a*c*t*c*c*a*g*a*t*g*U U U 

C iFQ 77 HPRT#1 5-10-5 gapPS 3x

U A G G a*c*t*c*

a*g*a*t*g*U U U 

C iFQ 78 HPRT#1 5-10-5 LNA PS A*T*A*G*G*a*c*t*c*c*a*g*a*t*g*T*T*T*C*CUppercase = 2′OMe RNA Lowercase = DNA Uppercase with underscore = LNA″*″ = phosphorothioate linkage ″

″ = insertion of napthyl-azo modifier (iFQ)

HeLa Cell Culture, Transfections, and RT-qPCR Methods.

HeLa cells were split into 48-well plates and were transfected the nextday at ˜60% confluency in serum-free Dulbecco's Modified Eagle Medium(Invitrogen, Carlsbad, Calif.) using TriFECTin® (Integrated DNATechnologies, Coralville, Iowa) at a concentration of 2% (1 μL per 50 μLOptiMEM® I) (Invitrogen, Carlsbad, Calif.) with ASOs at the indicatedconcentrations. All transfections were performed in triplicate. After 6hours, media was exchanged with Dulbecco's Modified Eagle Mediumcontaining 10% fetal bovine serum. RNA was prepared 24 hours aftertransfection using the SV96 Total RNA Isolation Kit (Promega, Madison,Wis.). cDNA was synthesized using 150 ng total RNA with SuperScript™-IIReverse Transcriptase (Invitrogen, Carlsbad, Calif.) per themanufacturer's instructions using both random hexamer and oligo-dTpriming. Transfection experiments were all performed a minimum of threetimes.

Quantitative real-time PCR was performed using 10 ng cDNA per 10 μLreaction with Immolase™ DNA Polymerase (Bioline, Randolph, Mass.), 200nM primers, and 200 nM probe. Hypoxanthine phosphoribosyltransferase 1(HPRT1) (GenBank Acc. No. NM_000194) specific primers were:

HPRT-For (SEQ ID NO: 79) 5′ GACTTTGCTTTCCTTGGTCAGGCA, HPRT-Rev(SEQ ID NO: 80) 5′ GGCTTATATCCAACACTTCGTGGG, and probe HPRT-P(SEQ ID NO: 81) 5′ MAX-ATGGTCAAGGTCGCAAGCTTGCTGGT-IowaBlackFQ (IBFQ)and were normalized to levels of an internal control gene, human acidicribosomal phosphoprotein P0 (RPLP0) (GenBank Acc. No. NM_001002), whichwas measured in a multiplexed reaction using primers:

RPLP0-For (SEQ ID NO: 82) 5′ GGCGACCTGGAAGTCCAACT, RPLP0-Rev(SEQ ID NO: 83) 5′ CCATCAGCACCACAGCCTTC, and probe RPLP0-P(SEQ ID NO: 84) 5′ FAM-ATCTGCTGCATCTGCTTGGAGCCCA-IBFQ(Bieche et al., 2000, Clin. Cancer Res. 6(2): 452-59). Cyclingconditions employed were: 95° C. for 10 minutes followed by 40 cycles of2-step PCR with 95° C. for 15 seconds and 60° C. for 1 minute. PCR andfluorescence measurements were done using an ABI Prism™ 7900 SequenceDetector (Applied Biosystems Inc., Foster City, Calif.). All reactionswere performed in triplicate. Expression data were normalized. Copynumber standards were multiplexed using linearized cloned amplicons forboth the HPRT and RPLP0 assays. Unknowns were extrapolated againststandards to establish absolute quantitative measurements.

Results.

ASOs were transfected into HeLa cells at 1 nM, 5 nM, and 20 nMconcentrations. RNA was prepared 24 hours post transfection, convertedto cDNA, and HPRT expression levels were measured using qPCR. Resultsare shown in FIG. 5 for the set of anti-HPRT ASOs made from DNA bases.Unmodified single-stranded DNA oligos are rapidly degraded byexonucleases and endonucleases. No knockdown of HPRT was observed usingthis design (HPRT DNA), presumably due to rapid degradation of theunprotected compound. ASOs with a single iFQ modification near the3′-end (HPRT DNA 3′ iFQ), a single iFQ modification near the 5′-end(HPRT DNA 5′ iFQ), two iFQ modifications inserted near both ends (HPRTDNA 5′+3′ iFQ), and three iFQ modifications inserted in the center andnear both ends (HPRT DNA 5′+I+3′ iFQ) were also tested and similarlyshowed no functional gene knockdown at the doses examined. Therefore,the addition of even up to three iFQ modifications does not providesufficient nuclease stabilization to permit otherwise unmodified DNAoligos to function as antisense gene-knockdown agents.

The same series of oligonucleotides was synthesized havingphosphorothioate (PS) internucleotide bonds throughout the sequence(except where the phosphate connects to an iFQ modifier). Historically,DNA-PS oligos were among the first effective antisense compoundsstudied. This modification increases nuclease stability; however, italso lowers binding affinity (T_(m)) and as a result this so-called“first generation” antisense chemistry usually shows relatively lowpotency. The “DNA-PS” ASO reduced HPRT levels by 50% at 20 nMconcentration; however, no reduction in HPRT levels was observed atlower doses. Addition of the iFQ modification, which increases bindingaffinity and blocks exonuclease action, improved function of the DNA-PSASOs. The “DNA-PS 5′+I+3′ iFQ” compound showed the best results withinthis series, with HPRT knockdown of 70% at 20 nM and 40% at 5 nMobserved (FIG. 5).

A second set of ASOs was synthesized using a chimeric “5-10-5 gapmer”design where five base end domains were made of 2′OMe RNA and a centralten base RNase H active domain were made of DNA. Oligonucleotides hadzero, one, two, or three iFQ modifiers inserted at the same positions asthe DNA ASOs in FIG. 5. These oligonucleotides were transfected intoHeLa cells as before and HPRT mRNA levels were examined 24 hourspost-transfection. Results are shown in FIG. 6. The three gapmer ASOswith a phosphodiester DNA central domain showed no activity in reducingHPRT mRNA levels, regardless of whether the sequence was modified withthe iFQ group or not (“5-10-5”, 5-10-5 2x iFQ” and “5-10-5 3x iFQ”). Thesame sequence fully PS modified (“5-10-5-PS”) showed good potency with75% knockdown of HPRT at 20 nM concentration. The addition of two iFQgroups near the ends of this design (“5-10-5-PS 2xiFQ”) showed the bestpotency of this set, with >90% knockdown of HPRT at 20 nM and >70%knockdown at 5 nM concentration.

Although 2′OMe RNA is somewhat resistant to endonuclease attack, gapmerASOs of this design are usually made with full PS modification toprevent exonuclease degradation. Consistent with this idea, an ASO withthe DNA domain protected by PS internucleotide linkages but havingphosphodiester bonds in the 2′OMe flanking domains showed no geneknockdown activity (“5-10-5 gapPS”). Use of the iFQ modification at theends, however, permits use of this new design by providing protectionfrom exonuclease attack; this new design should also increase bindingaffinity and lower toxicity by reducing PS content. This strategy waseffective and the ASO (“5-10-5 gapPS 2×iFQ”) showed knockdown of HPRTlevels by >90% at 20 nM and by >70% at 5 nM. Potency was very similar tothe full PS modified ASO. This design is expected to have reducedtoxicity; however, toxicity is not easily tested in this system as HeLacells are tolerant to fairly high doses of PS modified oligonucleotides.Benefit from reduced PS content will be better appreciated in vivo.

Although it did not increase functional potency, addition of a thirdcentrally placed iFQ group (“5-10-5 gapPS-3x-iFQ”) was compatible withgene knockdown in this RNase H active antisense design. It is generallyaccepted that maximal activity of RNase H active ASOs requires a DNAdomain having at least 8 uninterrupted DNA residues. It was unexpectedthat the 3xiFQ design (where the 10 base DNA domain is interrupted by acentral iFQ group) would work without reducing potency compared with the2xiFQ design (where the 10 base DNA domain is continuous). It ispossible that unique properties of the iFQ group allow its insertion toremain compatible with RNase H activity, possibly due to the samepostulated base stacking interactions that result in increased T_(m) inthese compounds.

The most potent antisense design in current use are LNA-modifiedgapmers, where very strong T_(m) enhancing LNA modifications are used inthe flanking domains in place of the 2′OMe RNA bases used in the presentexample. While potent, this design is expensive and can show significanttoxicity in certain contexts. The same anti-HPRT sequence was made as anLNA 5-10-5 gapmer (fully PS modified). As expected, this compound showedthe highest relative potency of any of the ASOs tested (“5-10-5 LNA PS”)but the observed potency was only marginally higher than the best of theiFQ compositions (“5-10-5-PS 2×iFQ”). The very high binding affinity LNAreagents usually result in decreased specificity, so use of the iFQdesigns of the present invention may show improved specificity at asmall cost in potency.

EXAMPLE 6

This example demonstrates use of the iFQ modification in RNA duplexeswith application in suppressing gene expression via an RNAi mechanism ofaction.

The use of double-stranded RNA (dsRNA) to trigger gene suppression viaRNA interference (RNAi) is a well-described technique. Synthetic dsRNAsthat mimic natural cellular products (small interfering RNAs, or siRNAs)are usually 21 bases long with a central 19 base duplex domain with2-base 3′-overhangs. Alternatively, slightly larger syntheticoligonucleotides can be used that are substrates for the cytoplasmicnuclease Dicer, which processes these species into 21-mer siRNAs.Typically these reagents are asymmetric and have a 25 base top (Sensestrand, “S”) and a 27 base bottom strand (Antisense strand, “AS”) with asingle 2-base 3′-overhang on the AS strand. These longer siRNAs arecalled Dicer-substrate siRNAs, or DsiRNAs. Although dsRNA is far morestable to nuclease attack than single-stranded RNA (ssRNA), degradationof the synthetic siRNAs can significantly limit potency of thecompounds, especially when used in vivo. Incorporation of chemicalmodifications, such as 2′OMe RNA, 2′F RNA, or LNA bases, improvesnuclease stability and can improve function of the siRNA. Selectiveplacement of nuclease-resistant phosphorothioate bonds (PS) can alsohelp stabilize the siRNA, especially when used near the terminal3′-internucleotide linkages. Unfortunately, careful placement ofmodified groups is essential as extensive chemical modification usuallylowers functional potency of the compound even though nucleasestabilization has been achieved, probably through disrupting interactionof the RNA duplex with key protein mediators of RNAi, like Dicer orAgo2.

The present example demonstrates that the iFQ modifier can be introducedinto DsiRNAs. Like other chemical modifiers, iFQ insertion can lead toincreased potency, decreased potency, or no change in potency dependingupon placement.

Oligonucleotide Synthesis and Purification.

RNA and modified RNA oligonucleotides were synthesized using solid phasephosphoramidite chemistry, deprotected and desalted according to routinetechniques (Caruthers et al., 1992). The oligomers were purified usingion-exchange high performance liquid chromatography (IE-HPLC) and werehandled under RNase-free conditions. All RNA oligonucleotides wereprepared as a sodium salt. The purity of each oligomer was determined bycapillary electrophoresis (CE) carried out on a Beckman P/ACE MDQ system(Beckman Coulter, Inc., Fullerton, Calif.). All single-strand oligomerswere at least 85% pure. Electrospray-ionization liquid chromatographymass spectrometry (ESI-LCMS) of the oligonucleotides was conducted usingan Oligo HTCS system (Novatia, Princeton, N.J.), which consisted ofThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processingsoftware, and Paradigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.).Protocols recommended by the manufacturers were followed. Experimentalmolar masses for all single-strand oligomers were within 1.5 g/mol ofexpected molar mass. These results confirm identity of the oligomers.

Duplexes were formed by mixing equal molar amounts of the top and bottomstrands in 30 mM Hepes, pH 7.5, 100 mM potassium acetate, heating at 95°C. for 2 minutes, then cooling to room temperature. Table 10 lists theduplexes synthesized for Example 6.

TABLE 10Synthetic RNA duplexes employed in Example 6 (anti-HPRT DsiRNAs) SEQ IDNO: Name Sequence 85 NC1 Negative Control 5′CGUUAAUCGCGUAUAAUACGCGUat 3′ S 86 3′ CAGCAAUUAGCGCAUAUUAUGCGCAUA 5′ AS87 HPRT unmod 5′ GCCAGACUUUGUUGGAUUUGAAAtt 3′ S 88 3′UUCGGUCUGAAACAACCUAAACUUUAA 5′ AS 89 HPRT iFQ v1 5′GCCAGACUUUGUUGGAUUUGAAAtt 3′ S 90 3′

UCGGUCUGAAACAACCUAAACUUUAA 5′ AS 91 HPRT iFQ v2 5′

CCAGACUUUGUUGGAUUUGAAAtt 3′ S 92 3′ UUC GGUCUGAAACAACCUAAACUUUAA 5′ AS93 HPRT iFQ v3 5′

CCAGACUUUGUUGGAUUUGAAA

t 3′ S 94 3′ UUC GGUCUGAAACAACCUAAACUUUAA 5′ AS 95 HPRT iFQ v4 5′G CCAGACUUUGUUGGAUUUGAAAtt 3′ S 96 3′ UU

GGUCUGAAACAACCUAAACUUUAA 5′ AS 97 HPRT iFQ v5 5′G CCAGACUUUGUUGGAUUUGAAAtt 3′ S 98 3′ UU

GGUCUGAAACAACCUAAACUUU

A 5′ AS 99 HPRT iFQ v6 5′

CCAGACUUUGUUGGAUUUGAAA

t 3′ S 100 3′ UU

GGUCUGAAACAACCUAAACUUUA A 5′ AS 101 HPRT iFQ v7 5′

CCAGACUUUGUUGGAUUUGAAA

t 3′ S 102 3′ UU

GGUCUGAAACAACCUAAACUUU

A 5′ AS Uppercase = RNA Lowercase = DNA ″

″ = insertion of napthyl-azo modifier (iFQ) Note: gaps have beenintroduced in sequences for the purpose of alignment only and do notrepresent any modification to sequence.

HeLa Cell Culture, Transfections, and RT-qPCR Methods.

HeLa cells were transfected in “reverse format” at ˜60% confluency(Invitrogen, Carlsbad, Calif.) using 1 μL Lipofectamine™ RNAiMAX per 50μL OptiMEM™ I (Invitrogen, Carlsbad, Calif.) with RNA duplexes at theindicated concentrations. All transfections were performed intriplicate. RNA was prepared 24 hours after transfection using the SV96Total RNA Isolation Kit (Promega, Madison, Wis.); cDNA was synthesizedusing 150 ng total RNA with SuperScript™-II Reverse Transcriptase(Invitrogen, Carlsbad, Calif.) per the manufacturer's instructions usingboth random hexamer and oligo-dT priming.

Quantitative real-time PCR reactions were done using 10 ng cDNA per 10μL reaction, Immolase™ DNA Polymerase (Bioline, Randolph, Mass.), 500 nMprimers, and 250 nM probe. Hypoxanthine phosphoribosyltransferase 1(HPRT1) (GenBank Acc, No. NM_000194) specific primers were:

HPRT-For (SEQ ID NO: 79) 5′ GACTTTGCTTTCCTTGGTCAGGCA, HPRT-Rev(SEQ ID NO: 80) 5′ GGCTTATATCCAACACTTCGTGGG, and probe HPRT-P(SEQ ID NO: 81) 5′ FAM-ATGGTCAAGGTCGCAAGCTTGCTGGT-IowaBlackFQ (IBFQ).Cycling conditions employed were: 95° C. for 10 minutes followed by 40cycles of 2-step PCR with 95° C. for 15 seconds and 60° C. for 1 minute.PCR and fluorescence measurements were done using an ABI Prism™ 7900Sequence Detector (Applied Biosystems Inc., Foster City, Calif.). Alldata points were performed in triplicate. Expression data werenormalized to levels of an internal control gene, human splicing factor,arginine/serine-rich 9 (SFRS9) (GenBank Acc. No. NM_003769), which wasmeasured in a multiplexed reaction using primers:

SFRS9-For (SEQ ID NO: 103) 5′ TGTGCAGAAGGATGGAGT, SFRS9-Rev(SEQ ID NO: 104) 5′ CTGGTGCTTCTCTCAGGATA, and probe SFRS9-P(SEQ ID NO: 105) 5′ MAX-TGGAATATGCCCTGCGTAAACTGGA-IBFQ,setting the baseline to cells transfected with a scrambled negativecontrol RNA duplex (NC1). Copy number standards were run in parallelusing linearized cloned amplicons for both the HPRT and SFRS9 assays.Unknowns were extrapolated against standards to establish absolutequantitative measurements.

Results.

The anti-HPRT DsiRNA employed in the present study is extremely potentand typically shows detectable knockdown of target mRNA at low picomolarlevels. Consistent with this expectation, the unmodified duplex reducedHPRT levels by ˜40% at a 10 μM dose at 24 hours post-transfection inHeLa cells. A series of modified duplexes containing the iFQ grouppositioned at various locations in the S strand, AS strand, or both weresimilarly transfected into HeLa cells and HPRT mRNA levels were measured24 hour post-transfection. Results are shown in FIG. 7.

Placing the iFQ group near the 3′-end of the AS strand was welltolerated; insertion between bases 1 and 2 from the 3′-end (in thesingle-stranded 3′-overhang domain) (duplex HPRT iFQ v1) or betweenbases 3 and 4 from the 3′-end (at the start of the duplex domain)(duplex HPRT iFQ v4) showed similar potency to the unmodified duplex.Placing the iFQ group near the 5′-end of the S strand was similarly welltolerated (duplex HPRT iFQ v2) as was placing the iFQ group near bothends of the S strand (duplex HPRT iFQ v3). In contrast, duplexes havingan iFQ group near the 5′-end of the AS strand showed reduced potency(duplexes HPRT iFQ v5 and v7), so modification at this position shouldbe avoided.

Within the error of the system studied, the iFQ modified and unmodifiedduplexes showed similar potency (except for those duplexes modified atthe 5′-end of the AS strand, as noted above). Benefit from the iFQ groupis most likely to be evident in settings where nuclease stabilization isneeded, which is not appreciated in the present in vitro system, butbased on the results of Examples 1 and 2, greater benefit would beexpected from use of this modification when used in vivo where exposureto serum nucleases is more problematic.

EXAMPLE 7

This example demonstrates decreased cellular toxicity from lipidtransfected internal napthyl-azo-containing oligomers compared withother compounds.

Toxicity from chemical modification of synthetic oligomers can beproblematic as it can give unwanted side effects, cause unreliableresults, and limit therapeutic utility of the oligomer. Cellular deathcan result from toxic chemical modifications by inducing necrosis orapoptosis. Toxicity was ascertained with oligomers containing anon-targeting, negative control (“NC1”) sequence using chemicalmodification patterns employed in the AMOs examined in Examples 3 and 4(see Table 11). Generalized cytotoxicity (from necrosis and/orapoptosis) was measured by quantifying the relative number of live anddead cells after treatment with the chemically modified oligomers, whilecytotoxicity resulting from the induction of the apoptotic pathway wasdetermined by measuring the levels of caspase-3 and -7 after oligomertreatment.

Oligonucleotide Synthesis and Preparation.

DNA oligonucleotides were synthesized using solid phase phosphoramiditechemistry, deprotected and desalted on NAP-5 columns (Amersham PharmaciaBiotech, Piscataway, N.J.) according to routine techniques (Caruthers etal., 1992). The oligomers were purified using reversed-phase highperformance liquid chromatography (RP-HPLC). The purity of each oligomerwas determined by capillary electrophoresis (CE) carried out on aBeckman P/ACE MDQ system (Beckman Coulter, Inc., Fullerton, Calif.). Allsingle-strand oligomers were at least 90% pure. Electrospray-ionizationliquid chromatography mass spectrometry (ESI-LCMS) of theoligonucleotides was conducted using an Oligo HTCS system (Novatia,Princeton, N.J.), which consisted of ThermoFinnigan TSQ7000, Xcaliburdata system, ProMass data processing software, and Paradigm MS4™ HPLC(Michrom BioResources, Auburn, Calif.). Protocols recommended by themanufacturers were followed. Experimental molar masses for allsingle-strand oligomers were within 1.5 g/mol of expected molar mass.These results confirm identity of the oligomers.

TABLE 11  Synthetic oligomers employed in Example 7 (NC1 AMOs) SEQ IDNO: Name Sequence 106 2′OMe 5′ + 3′ iFQG_(z)C G U A U U A U A G C C G A U U A A C G_(z)A 107 2′OMe PSendsG*C*G*U A U U A U A G C C G A U U A A*C*G*A 108 DNA/PSg*c*g*t*a*t*t*a*t*a*g*c*c*g*a*t*t*a*a*c*g*a 109 DNA/LNA POg C g t A t t A t a G c c G a t T a a C g a 110 DNA/LNA PSg*C*g*t*A*t*t*A*t*a*G*c*c*G*a*t*T*a*a*C*g*a 111 2′OMe/LNA POG C G T A T T A T A G C C G A T T A A C G A 112 2′OMe/LNA PSG*C*G*T*A*T*T*A*T*A*G*C*C*G*A*T*T*A*A*C*G*A Uppercase = 2′OMe RNALowercase = DNA Uppercase with underscore = LNA “*” = phosphorothioatelinkage “_(z)” = napthyl-azo modifier (iFQ)

Cell Culture, Transfections, and Luciferase Assays.

HeLa cells were plated in 48-well plates in DMEM containing 10% FBS toachieve 90% confluency the next day. The following morning, NC1 AMOswere transfected at 100 nM or 50 nM concentrations in triplicate wellsin two sets (one for measuring general cytotoxicity, one for measuringapoptosis induction) with 1 μl TriFECTin® (Integrated DNA Technologies)per well in DMEM containing 10% FBS. An apoptosis-inducing agent,Staurosporine (1 mM in DMSO), was incubated at 1 μM on the cells for 24hours as a positive control. After 24 hours of NC 1 AMO treatment, thefirst set of cells was analyzed for viability using the MultiTox-GloMultiplex Cytotoxicity Assay (Promega, Madison, Wis.) with thepeptide-substrate GF-AFC (glycyl-phenylalanylaminofluorocoumarin), whichgenerates a fluorescence signal upon cleavage by a “live-cell” specificprotease, measured at 405 nm_(Ex)/505 nm_(Em) in a SpectraFluorMicroplate Reader (Tecan Group Ltd, Männedorf, Switzerland). Continuingto use the MultiTox-Glo Multiplex Cytotoxicity Assay, the same cellswere subsequently analyzed for cytotoxicity by detecting a “dead-cell”protease activity in a luciferase-based assay measured on a GloMax® 96Microplate Luminometer (Promega) per the manufacturer's recommendations.To assess cytotoxicity derived from induction of the apoptosis pathway,the Caspase-Glo® 3/7 Assay (Promega) was performed with the second setof cells to measure caspase-3 and -7 levels according to themanufacturer's recommendations on a GloMax® 96 Microplate Luminometer(Promega).

Results. For the cytotoxicity analysis graphed in FIG. 8, data ispresented as a ratio of live/dead cells as calculated from the abundanceof “live-cell” and “dead-cell” proteases described above. The ratio oflive/dead cells serves as an internal normalizing control providing dataindependent of cell number, and a reduction of live/dead cellscorrelates with cytotoxicity. The “2′OMe 5′+3′ iFQ” and “2′OMe PSends”compounds are the least toxic oligomers and there is minimal toxicityeven at the high 100 nM dose. The “DNA/PS” oligomer, which is entirelycomprised of PS linkages, shows substantial cell death at both dosessuggesting that PS modification is toxic to the cells. When LNA basesare incorporated into the NC1 AMO, such as in the “DNA/LNA PO” oligomer,cell death is seen at the high 100 nM dose suggesting that LNAmodification is toxic to the cells. Importantly, additive cell death isseen after combining these two chemistries in the “DNA/LNA PS” oligomer,demonstrating toxicity which also correlates with the dysmorphic,unhealthy cells seen during the visual analysis at the time the assaywas performed. Substituting DNA with 2° OMe bases in the “2° OMe/LNA PO”and “2′OMe/LNA PS” NC1 AMOs reduces toxicity compared with their DNAcounterparts; however, cell death is still seen at the 100 nM dose.

In parallel, HeLa cells treated with the NC1 AMOs were assessed forapoptosis induction by evaluating the levels of caspase-3 and -7 in aluciferase-based assay (FIG. 9). Luminescence is proportional to theabundance of the apoptosis effectors and an increase in RLUs correlateswith an increase in apoptosis. The data in FIG. 9 mirrors thecytotoxicity profiles from the NC1 AMOs assayed in FIG. 8. The NC1 AMOsthat do not trigger apoptosis are the “2′OMe 5′+3′ iFQ” and “2′OMePSends” compounds. Both extensive PS modification (“DNA/PS”) andincorporation of LNA bases (“DNA/LNA PO”) induce apoptosis, while anadditive effect is seen when these two chemistries are combined(“DNA/LNA PS”). Again, substitution of DNA bases for 2′OMe bases(“2′OMe/LNA PO” and “2′OMe/LNA PS”) reduces apoptosis induction.However, the “2′OMe/LNA PS” still demonstrates apoptosis induction atthe 100 nM dose.

This cytotoxicity profiling analysis clearly exemplifies that certainchemical modification strategies can be detrimental to cell viability.The “2′OMe 5′+3′ iFQ” AMO and the “DNA/LNA PS” AMOs, which demonstratedsimilar high potency in Example 3, have significantly different toxicityprofiles. The “2′OMe iFQ” oligomer was non-toxic in this system, and the“DNA/LNA PS” oligomer caused substantial cell death in FIG. 8 and wasshown to induce apoptosis in FIG. 9. These data confirm the superiorityof the “2′OMe 5′+3′ iFQ” AMO when compared to other standard AMOs withcomparable potency (Example 3), increased specificity (Example 4), andreduced toxicity.

EXAMPLE 8

This example demonstrates that the magnitude of T_(m) enhancement (i.e.,stability) derived from use of the internal napthyl-azo modificationvaries with the nearest-neighbor base context.

It is well established that the binding affinity (or T_(m)) of a nucleicacid duplex varies with base composition. Further, the identity of theflanking bases (i.e., “nearest neighbors”) directly influences T_(m)(Santa Lucia Proc. Natl. Acad. Sci. USA, Vol. 95, pp. 1460-1465, 1998).Example 1 demonstrated positive T_(m) effects when the napthyl-azomodifier was incorporated in a short 10mer DNA oligomer duplexed with aDNA complement. The present example demonstrates the T_(m) effects ofthe napthyl-azo modifier when incorporated in 22-mer 2′OMe oligomersduplexed with RNA complements.

Specifically, this study examines the T_(m) effects caused by insertionof the napthyl-azo modifier between 5′- or 3′-terminal 2′OMe residues of22mer 2′OMe oligomers when duplexed with a perfect match complementaryRNA target. This artificial system mimics behavior of a steric blockingASO binding a miRNA target (e.g., an AMO). Nearest neighbor base effectswere studied for all 16 possible dinucleotide pairs. The effects of thenapthyl-azo modifier placed externally at the 5′-end or 3′-end of the2′OMe oligomer were also studied.

Oligonucleotide Synthesis and Preparation.

2′OMe and RNA oligonucleotides were synthesized using solid phasephosphoramidite chemistry, deprotected and desalted on NAP-5 columns(Amersham Pharmacia Biotech, Piscataway, N.J.) according to routinetechniques (Caruthers et al., 1992, Methods Enzymol. 211: 3-20). Theoligomers were purified using reversed-phase high performance liquidchromatography (RP-HPLC). The purity of each oligomer was determined bycapillary electrophoresis (CE) carried out on a Beckman P/ACE MDQ system(Beckman Coulter, Inc., Fullerton, Calif.). All single-strand oligomerswere at least 90% pure. Electrospray-ionization liquid chromatographymass spectrometry (ESI-LCMS) of the oligonucleotides was conducted usingan Oligo HTCS system (Novatia, Princeton, N.J.), which consisted ofThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processingsoftware, and Paradigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.).Protocols recommended by the manufacturers were followed. Experimentalmolar masses for all single-strand oligomers were within 1.5 g/mol ofexpected molar mass. These results confirm identity of the oligomers.

Preparation of Samples.

Melting experiments were carried out in buffer containing 3.87 mMNaH₂PO₄, 6.13 mM Na₂HPO₄, 1 mM Na₂EDTA, and 130 mM NaCl, i.e., close tophysiologic saline. 1 M NaOH was used to titrate each solution to pH7.0. Total sodium concentrations were estimated to be 150 mM. The DNAsamples were thoroughly dialyzed against melting buffer in a 28-wellMicrodialysis System (Life Technologies, Carlsbad, Calif.) following themanufacturer's recommended protocol. Concentrations of oligomers wereestimated from the samples' UV absorbance at 260 nm in aspectrophotometer (Beckman Coulter, Inc., Fullerton, Calif.), usingextinction coefficients for each oligonucleotide that were estimatedusing the nearest neighbor model for calculating extinction coefficients(see Warshaw et al., 1966, J. Mol. Biol. 20(1): 29-38).

Internal Modifications Studied.

The napthyl-azo compound (Formula 3, Integrated DNA Technologies, Inc.,sometimes referred to as “iFQ” or “ZEN” in this disclosure), wasintroduced into 2′OMe oligonucleotides using phosphoramidite reagents atthe time of synthesis.

In some of the oligomers, the group was placed as an insertion betweenthe 5′- or 3′-terminal bases (iFQ); in other cases, the group was placedat the 5′- or 3′-end and was not inserted between bases (FQ). Extinctioncoefficients at 260 nm of iFQ were estimated to be 13340.

Measurement of Melting Curves.

Oligomer concentrations were measured at least twice for each sample. Ifthe estimated concentrations for any sample differed more than 4%, theresults were discarded and new absorbance measurements were performed.To prepare oligonucleotide duplexes, complementary 2′OMe:RNA oligomerswere mixed in 1:1 molar ratio, heated to 367 K (i.e., 94° C.) and slowlycooled to an ambient temperature. Each solution of duplex was dilutedwith melting buffer to a total concentration (C_(T)) of 2 μM.

Melting experiments were conducted on a single beam Beckman DU 650spectrophotometer (Beckman-Coulter) with a Micro T_(m) Analysisaccessory, a Beckman High Performance Peltier Controller (to regulatethe temperature), and 1 cm path-length cuvettes. Melt data were recordedusing a PC interfaced to the spectrophotometer. UV-absorbance values at268 nm wavelength were measured at 0.1 degree increments in thetemperature range from 383 to 368 K (i.e., 10-95° C.). Both heating(i.e., “denaturation”) and cooling (i.e., “renaturation”) transitioncurves were recorded in each sample at a controlled rate of temperaturechange (24.9±0.3° C. per hour). Sample temperatures were collected fromthe internal probe located inside the Peltier holder, and recorded witheach sample's UV-absorbance data. Melting profiles were also recordedfor samples of buffer alone (no oligonucleotide), and these “blank”profiles were digitally subtracted from melting curves of the DNAsamples. To minimize systematic errors, at least two melting curves werecollected for each sample in different cuvettes and in differentpositions within the Peltier holder.

Determination of Melting Temperatures.

To determine each sample's melting temperature, the melting profileswere analyzed using methods that have been described (see Doktycz etal., 1992, Biopolymers 32(7): 849-64; Owczarzy et al., 1997, Biopolymers44(3): 217-39; and Owczarzy, 2005, Biophys. Chem. 117(3): 207-15.).Briefly, the experimental data for each sample was smoothed, using adigital filter, to obtain a plot of the sample's UV-absorbance as afunction of its temperature. The fraction of single-strandedoligonucleotide molecules, 0, was then calculated from that plot. The“melting temperature” or “T_(m)” of a sample was defined as thetemperature where θ=0.5.

Results.

2′OMe oligomers modified with an internal napthyl-azo modifier placedbetween the terminal 5′-end residues or the terminal 3′-end residueswere duplexed with RNA targets to mimic complexation between an AMO anda target miRNA. Control duplexes without the napthyl-azo modifier werestudied in parallel. UV melt profiles were collected as described aboveunder physiologic conditions. T_(m) values were collected for theforward and reverse melt curves for three independent samples. Theaverage T_(m) values for the set are shown in Table 12. The T_(m) valueof the control duplex was subtracted from the T_(m) value of themodified duplex to yield a ΔT_(m) value, which represents the change instability seen with modification.

TABLE 12 T_(m) of napthyl-azo internally-modified 2′OMe AMOs duplexed with RNASEQ ID Dinucleotide No. Sequence pair studied T_(m)° C. ΔT_(m)° C. 1135′ UzCAACAUCAGUCUGAUAAGCUA 3′ 5′ UzC . . . 3′ 77.0 +4.0 114 3′A GUUGUAGUCAGACUAUUCGAU 5′ 115 5′ UCAACAUCAGUCUGAUAAGCUA 3′ 73.0 114 3′AGUUGUAGUCAGACUAUUCGAU 5′ 116 5′ UzGAACAUCAGUCUGAUAAGCUA 3′ 5′UzG . . . 3′ 76.5 +2.2 117 3′ A CUUGUAGUCAGACUAUUCGAU 5′ 118 5′UGAACAUCAGUCUGAUAAGCUA 3′ 74.3 117 3′ ACUUGUAGUCAGACUAUUCGAU 5′ 119 5′UzAAACAUCAGUCUGAUAAGCUA 3′ 5′ UzA . . . 3′ 73.5 +0.4 120 3′A UUUGUAGUCAGACUAUUCGAU 5′ 121 5′ UAAACAUCAGUCUGAUAAGCUA 3′ 73.1 120 3′AUUUGUAGUCAGACUAUUCGAU 5′ 122 5′ UzUAACAUCAGUCUGAUAAGCUA 3′ 5′UzU . . . 3′ 73.5 +1.3 123 3′ A AUUGUAGUCAGACUAUUCGAU 5′ 124 5′UUAACAUCAGUCUGAUAAGCUA 3′ 72.2 123 3′ AAUUGUAGUCAGACUAUUCGAU 5′ 125 5′CzCAACAUCAGUCUGAUAAGCUA 3′ 5′ CzC . . . 3′ 76.1 +1.3 126 3′G GUUGUAGUCAGACUAUUCGAU 5′ 127 5′ CCAACAUCAGUCUGAUAAGCUA 3′ 74.8 126 3′GGUUGUAGUCAGACUAUUCGAU 5′ 128 5′ CzGAACAUCAGUCUGAUAAGCUA 3′ 5′CzG . . . 3′ 76.7 +2.3 129 3′ G CUUGUAGUCAGACUAUUCGAU 5′ 130 5′CGAACAUCAGUCUGAUAAGCUA 3′ 74.4 129 3′ GCUUGUAGUCAGACUAUUCGAU 5′ 131 5′CzAAACAUCAGUCUGAUAAGCUA 3′ 5′ CzA . . . 3′ 73.5 +0.6 132 3′G UUUGUAGUCAGACUAUUCGAU 5′ 133 5′ CAAACAUCAGUCUGAUAAGCUA 3′ 72.9 132 3′GUUUGUAGUCAGACUAUUCGAU 5′ 134 5′ CzUAACAUCAGUCUGAUAAGCUA 3′ 5′CzU . . . 3′ 75.5 +3.3 135 3′ G AUUGUAGUCAGACUAUUCGAU 5′ 136 5′CUAACAUCAGUCUGAUAAGCUA 3′ 72.2 135 3′ GAUUGUAGUCAGACUAUUCGAU 5′ 137 5′GzCAACAUCAGUCUGAUAAGCUA 3′ 5′ GzC . . . 3′ 75.7 +0.2 138 3′C GUUGUAGUCAGACUAUUCGAU 5′ 139 5′ GCAACAUCAGUCUGAUAAGCUA 3′ 75.5 138 3′CGUUGUAGUCAGACUAUUCGAU 5′ 140 5′ GzGAACAUCAGUCUGAUAAGCUA 3′ 5′GzG. . . 3′ 75.6 +0.7 141 3′ C CUUGUAGUCAGACUAUUCGAU 5′ 142 5′GGAACAUCAGUCUGAUAAGCUA 3′ 74.9 141 3′ CCUUGUAGUCAGACUAUUCGAU 5′ 143 5′GzAAACAUCAGUCUGAUAAGCUA 3′ 5′ GzA . . . 3′ 73.2 +0.1 144 3′C UUUGUAGUCAGACUAUUCGAU 5′ 145 5′ GAAACAUCAGUCUGAUAAGCUA 3′ 73.1 144 3′CUUUGUAGUCAGACUAUUCGAU 5′ 146 5′ GzUAACAUCAGUCUGAUAAGCUA 3′ 5′GzU . . . 3′ 73.9 +1.7 147 3′ C AUUGUAGUCAGACUAUUCGAU 5′ 148 5′GUAACAUCAGUCUGAUAAGCUA 3′ 72.2 147 3′ CAUUGUAGUCAGACUAUUCGAU 5′ 149 5′AzCAACAUCAGUCUGAUAAGCUA 3′ 5′ AzC . . . 3′ 76.5 +3.4 150 3′U GUUGUAGUCAGACUAUUCGAU 5′ 151 5′ ACAACAUCAGUCUGAUAAGCUA 3′ 73.1 150 3′UGUUGUAGUCAGACUAUUCGAU 5′ 152 5′ AzGAACAUCAGUCUGAUAAGCUA 3′ 5′AzG . . . 3′ 75.7 +1.3 153 3′ U CUUGUAGUCAGACUAUUCGAU 5′ 154 5′AGAACAUCAGUCUGAUAAGCUA 3′ 74.4 153 3′ UCUUGUAGUCAGACUAUUCGAU 5′ 155 5′AzAAACAUCAGUCUGAUAAGCUA 3′ 5′ AzA . . . 3′ 73.3 +0.2 156 3′U UUUGUAGUCAGACUAUUCGAU 5′ 157 5′ AAAACAUCAGUCUGAUAAGCUA 3′ 73.1 156 3′UUUUGUAGUCAGACUAUUCGAU 5′ 158 5′ AzUAACAUCAGUCUGAUAAGCUA 3′ 5′AzU . . . 3′ 73.8 +1.1 159 3′ U AUUGUAGUCAGACUAUUCGAU 5′ 160 5′AUAACAUCAGUCUGAUAAGCUA 3′ 72.7 159 3′ UAUUGUAGUCAGACUAUUCGAU 5′ 161 5′UCAACAUCAGUCUGAUAAGCUzC 3′ 5′ UzC . . . 3′ 74.1 −0.3 162 3′AGUUGUAGUCAGACUAUUCGA G 5′ 163 5′ UCAACAUCAGUCUGAUAAGCUC 3′ 74.4 162 3′AGUUGUAGUCAGACUAUUCGAG 5′ 164 5′ UCAACAUCAGUCUGAUAAGCUzG 3′ 5′. . . UzG 3′ 73.6 −0.7 165 3′ AGUUGUAGUCAGACUAUUCGA C 5′ 166 5′UCAACAUCAGUCUGAUAAGCUG 3′ 74.3 165 3′ AGUUGUAGUCAGACUAUUCGAC 5′ 167 5′UCAACAUCAGUCUGAUAAGCUzA 3′ 5′ . . . UzA 3′ 73.8 +0.6 168 3′AGUUGUAGUCAGACUAUUCGA U 5′ 169 5′ UCAACAUCAGUCUGAUAAGCUA 3′ 73.2 168 3′AGUUGUAGUCAGACUAUUCGAU 5′ 170 5′ UCAACAUCAGUCUGAUAAGCUzU 3′ 5′. . . UzU 3′ 74.4 +0.4 171 3′ AGUUGUAGUCAGACUAUUCGA A 5′ 172 5′UCAACAUCAGUCUGAUAAGCUU 3′ 74.0 171 3′ AGUUGUAGUCAGACUAUUCGAA 5′ 173 5′UCAACAUCAGUCUGAUAAGCCzC 3′ 5′ . . . CzC 3′ 75.0 −1.7 174 3′AGUUGUAGUCAGACUAUUCGG G 5′ 175 5′ UCAACAUCAGUCUGAUAAGCCC 3′ 76.7 174 3′AGUUGUAGUCAGACUAUUCGGG 5′ 176 5′ UCAACAUCAGUCUGAUAAGCCzG 3′ 5′. . . CzG 3′ 74.4 −1.6 177 3′ AGUUGUAGUCAGACUAUUCGG C 5′ 178 5′UCAACAUCAGUCUGAUAAGCCG 3′ 76.0 177 3′ AGUUGUAGUCAGACUAUUCGGC 5′ 179 5′UCAACAUCAGUCUGAUAAGCCzA 3′ 5′ . . . CzA 3′ 74.6 0.0 180 3′AGUUGUAGUCAGACUAUUCGG U 5′ 181 5′ UCAACAUCAGUCUGAUAAGCCA 3′ 74.6 180 3′AGUUGUAGUCAGACUAUUCGGU 5′ 182 5′ UCAACAUCAGUCUGAUAAGCCzU 3′ 5′. . . CzU 3′ 75.0 +0.5 183 3′ AGUUGUAGUCAGACUAUUCGG A 5′ 184 5′UCAACAUCAGUCUGAUAAGCCU 3′ 74.5 183 3′ AGUUGUAGUCAGACUAUUCGGA 5′ 185 5′UCAACAUCAGUCUGAUAAGCGzC 3′ 5′ . . . GzC 3′ 76.8 +0.2 186 3′AGUUGUAGUCAGACUAUUCGC G 5′ 187 5′ UCAACAUCAGUCUGAUAAGCGC 3′ 76.6 186 3′AGUUGUAGUCAGACUAUUCGCG 5′ 188 5′ UCAACAUCAGUCUGAUAAGCGzG 3′ 5′. . . GzG 3′ 77.0 +0.1 189 3′ AGUUGUAGUCAGACUAUUCGC C 5′ 190 5′UCAACAUCAGUCUGAUAAGCGG 3′ 76.9 189 3′ AGUUGUAGUCAGACUAUUCGCC 5′ 191 5′UCAACAUCAGUCUGAUAAGCGzA 3′ 5′ . . . GzA 3′ 76.1 −0.2 192 3′AGUUGUAGUCAGACUAUUCGC U 5′ 193 5′ UCAACAUCAGUCUGAUAAGCGA 3′ 192 3′AGUUGUAGUCAGACUAUUCGCU 5′ 76.3 194 5′ UCAACAUCAGUCUGAUAAGCGzU 3′ 5′. . . GzU 3′ 76.3 +0.9 195 3′ AGUUGUAGUCAGACUAUUCGC A 5′ 196 5′UCAACAUCAGUCUGAUAAGCGU 3′ 75.4 195 3′ AGUUGUAGUCAGACUAUUCGCA 5′ 197 5′UCAACAUCAGUCUGAUAAGCAzC 3′ 5′ . . . AzC 3′ 73.7 −0.4 198 3′AGUUGUAGUCAGACUAUUCGU G 5′ 199 5′ UCAACAUCAGUCUGAUAAGCAC 3′ 74.1 198 3′AGUUGUAGUCAGACUAUUCGUG 5′ 200 5′ UCAACAUCAGUCUGAUAAGCAzG 3′ 5′. . . AzG 3′ 72.9 −0.9 201 3′ AGUUGUAGUCAGACUAUUCGU C 5′ 202 5′UCAACAUCAGUCUGAUAAGCAG 3′ 73.8 201 3′ AGUUGUAGUCAGACUAUUCGUC 5′ 203 5′UCAACAUCAGUCUGAUAAGCAzA 3′ 5′ . . . AzA 3′ 73.6 +0.1 204 3′AGUUGUAGUCAGACUAUUCGU U 5′ 205 5′ UCAACAUCAGUCUGAUAAGCAA 3′ 73.5 204 3′AGUUGUAGUCAGACUAUUCGUU 5′ 206 5′ UCAACAUCAGUCUGAUAAGCAzU 3′ 5′. . . AzU 3′ 73.1 −0.2 207 3′ AGUUGUAGUCAGACUAUUCGU A 5′ 208 5′UCAACAUCAGUCUGAUAAGCAU 3′ 73.3 207 3′ AGUUGUAGUCAGACUAUUCGUA 5′ Topstrands are 2′OMe RNA (bold font) and are shown in 5′-3′ orientation.Bottom strands are RNA (standard font) and are shown in 3′-5′orientation. “z” indicates the iFQ napthyl-azo modifier.

For all 16 dinucleotide pairs, insertion of the napthyl-azo modifierbetween the 5′-most and adjacent base increased stability of the2′OMe:RNA duplex. Values range from +4.0° C. to +0.1° C., varying withnearest neighbor pairs. The average T_(m) value was increased by +1.5°C. with iFQ modification. Modification of the 3′-most and adjacent basealtered T_(m). Effects range from T_(m) stabilizing at +0.9° C. to T_(m)destabilizing at −1.7° C., with an average effect of −0.2° C. Thus theprecise T_(m) effects of insertion of the napthyl-azo modifier variedwith sequence context and whether the modification was at the 5′-end or3′-end of the oligomer.

The effects of terminal modification were studied next, where thenapthyl-azo modifier was placed at either the 5′-end or 3′-end of the2′OMe oligomer (not between terminal residues as before). The stabilityof 2′OMe:RNA duplexes was studied in an identical fashion for each ofthe 4 possible bases on each end of the modified strand. Results areshown in Table 13.

TABLE 13  T_(m) of napthyl-azo end-modified 2′OMe AMOs duplexed with RNASEQ ID End-base No. Sequence studied T_(m)° C. ΔT_(m)° C. 209 5′zUCAACAUCAGUCUGAUAAGCUA 3′ 5′ zU . . . 3′ 76.1 +2.9 114 3′AGUUGUAGUCAGACUAUUCGAU 5′ 115 5′ UCAACAUCAGUCUGAUAAGCUA 3′ 73.2 114 3′AGUUGUAGUCAGACUAUUCGAU 5′ 210 5′ zCCAACAUCAGUCUGAUAAGCUA 3′ 5′zC . . . 3′ 78.4 +3.4 126 3′ GGUUGUAGUCAGACUAUUCGAU 5′ 127 5′CCAACAUCAGUCUGAUAAGCUA 3′ 75.0 126 3′ GGUUGUAGUCAGACUAUUCGAU 5′ 211 5′zGCAACAUCAGUCUGAUAAGCUA 3′ 5′ zG . . . 3′ 78.3 +3.3 138 3′CGUUGUAGUCAGACUAUUCGAU 5′ 139 5′ GCAACAUCAGUCUGAUAAGCUA 3′ 75.0 138 3′CGUUGUAGUCAGACUAUUCGAU 5′ 212 5′ zACAACAUCAGUCUGAUAAGCUA 3′ 5′zA . . . 3′ 75.1 +1.9 150 3′ UGUUGUAGUCAGACUAUUCGAU 5′ 151 5′ACAACAUCAGUCUGAUAAGCUA 3′ 73.2 150 3′ UGUUGUAGUCAGACUAUUCGAU 5′ 213 5′UCAACAUCAGUCUGAUAAGCUCz 3′ 5′ . . . Cz 3′ 73.5 −1.2 162 3′AGUUGUAGUCAGACUAUUCGAG 5′ 163 5′ UCAACAUCAGUCUGAUAAGCUC 3′ 74.7 162 3′AGUUGUAGUCAGACUAUUCGAG 5′ 214 5′ UCAACAUCAGUCUGAUAAGCUGz 3′ 5′. . . Gz 3′ 74.1 −1.0 165 3′ AGUUGUAGUCAGACUAUUCGAC 5′ 166 5′UCAACAUCAGUCUGAUAAGCUG 3′ 75.1 165 3′ AGUUGUAGUCAGACUAUUCGAC 5′ 215 5′UCAACAUCAGUCUGAUAAGCUAz 3′ 5′ . . . Az 3′ 74.4 +1.2 168 3′AGUUGUAGUCAGACUAUUCGAU 5′ 169 5′ UCAACAUCAGUCUGAUAAGCUA 3′ 73.2 168 3′AGUUGUAGUCAGACUAUUCGAU 5′ 216 5′ UCAACAUCAGUCUGAUAAGCUUz 3′ 5′. . . Uz 3′ 74.7 +1.7 171 3′ AGUUGUAGUCAGACUAUUCGAA 5′ 172 5′UCAACAUCAGUCUGAUAAGCUU 3′ 73.0 171 3′ AGUUGUAGUCAGACUAUUCGAA 5′ Topstrands are 2′OMe RNA (bold font) and are shown in 5′-3′ orientation.Bottom strands are RNA (standard font) and are shown in 3′-5′orientation. “z” indicates the terminal FQ napthyl-azo modifier.

The effect of modification with the napthyl-azo group at the 5′-end of a2′OMe oligomer duplexed to RNA varied from +3.4° C. to +1.9° C.,averaging +2.9° C. for the 4 bases. The effect of modification with thenapthyl-azo group at the 3′-end of a 2′OMe oligomer duplexed to RNAvaried from +1.7° C. to −1.2° C., averaging +0.2° C. for the 4 bases.Thus terminal addition of the napthyl-azo modifier affects duplexstability in a similar fashion to internal modification. The magnitudeof the T_(m) effect varies with sequence. It is possible that functionalpotency of a modified antisense oligomer may vary slightly withplacement of the napthyl-azo modifier depending on sequence context.

EXAMPLE 9

This example demonstrates improved nuclease stability ofnapthyl-azo-modified 2′OMe oligomers. Stability of internalnapthyl-azo-modified DNA oligomers was demonstrated in Example 2. Thepresent example extends this study to examine the effects of thismodification on 2′OMe oligomers in serum and in cell extracts. The 2′OMeRNA backbone shows higher binding affinity to RNA targets than DNA andshows some intrinsic resistance to nuclease degradation which issignificantly improved through use of the napthyl-azo modifier.

Oligonucleotide Synthesis and Purification.

Oligonucleotides were synthesized using solid phase phosphoramiditechemistry, deprotected and desalted on NAP-5 columns (Amersham PharmaciaBiotech, Piscataway, N.J.) according to routine techniques (Caruthers etal., 1992). The oligomers were purified using reversed-phase highperformance liquid chromatography (RP-HPLC). The purity of each oligomerwas determined by capillary electrophoresis (CE) carried out on aBeckman P/ACE MDQ system (Beckman Coulter, Inc., Fullerton, Calif.). Allsingle-strand oligomers were at least 90% pure. Electrospray-ionizationliquid chromatography mass spectrometry (ESI-LCMS) of theoligonucleotides was conducted using an Oligo HTCS system (Novatia,Princeton, N.J.), which consisted of ThermoFinnigan TSQ7000, Xcaliburdata system, ProMass data processing software, and Paradigm MS4™ HPLC(Michrom BioResources, Auburn, Calif.). Protocols recommended by themanufacturers were followed. Experimental molar masses for allsingle-strand oligomers were within 1.5 g/mol of expected molar mass.These results confirm identity of the oligomers. The synthesizedoligonucleotides are listed in Table 14.

Nuclease Stability Assay Methods.

A male mouse was sacrificed via cervical dislocation. One gram of livertissue was placed into 10 ml of T-PER tissue protein extraction reagent(Pierce, Rockford, Ill.) containing a 1/100 volume cocktail of proteaseinhibitors comprising 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF),pepstatin-A, E-64, bestatin, leupeptin, and aprotinin (Sigma-Aldrich,St. Louis, Mo.). The liver-extraction reagent mixture was immediatelyhomogenized at 35,000 RPMs for 1 minute using a 10 mm stainless steelprobe on an Omni TH homogenizer (Omni International, Kennesaw, Ga.),followed by centrifugation at 10,000×g for 5 min. The supernatant wasstored at −80° C.

In a 70 μl total reaction volume, the oligomers (AMOs) were diluted to15 μM in PBS and incubated in 10% and 50% non-heat-inactivated fetalbovine serum (FBS), or 20% and 50% mouse liver protein extract at 37° C.for 0, 2, 6 or 24 hrs. Degradation reactions were stopped at each timepoint by adding an equal volume of 2× formamide gel loading buffer (90%formamide, 1×TBE, 0.025% w/v bromophenol blue and 0.025% w/v xylenecyanol) and immediately flash freezing on dry ice with subsequentstorage at −80° C. 200 pmoles (13.33 μl) of each reaction was heated to95° C. for 5 minutes and placed on ice for 2 min, loaded on 7M urea 20%polyacrylamide gels and electrophoresed at 30 mAs. Gels were stained for30 minutes in a methylene blue solution (0.02% w/v methylene blue in0.1×TBE), destained in several washes of H2O for 2 hrs and images weregenerated with an HP Scanjet 4850 Photo Scanner (Hewlett-PackardCompany, Palo Alto, Calif.).

A 15 μl (225 pmoles) aliquot of each degradation reaction in both 10%FBS and 20% mouse liver protein extract at the 24 hr time point wasanalyzed by electrospray ionization liquid chromatography massspectrometry (ESI-LC-MS) for evaluation of degradation products. The 20%mouse liver protein extract reactions were incubated with 200 μg/ml ofProteinase K (Sigma-Aldrich) at 37° C. for 1 hour prior to massspectometry. The 10% FBS and Proteinase K-digested 20% mouse liverprotein extract treated oligomers were extracted with an equal volume ofphenol:chloroform:isoamyl alcohol 25:24:1 (Sigma-Aldrich) and ethanolprecipitated (3 μl of 10 μg/μ1 glycogen, 1/10 vol 3M Na+ Acetate pH 5.2,2.5 vol cold EtOH). Pellets were re-suspended in 60 μl H2O and theentire sample was analyzed by ESI-LC-MS.

Results.

Oligomers employed in the nuclease stability studies are listed in Table14. The sequences are complementary to miR-21 and represent chemicalvariants of miR-21 AMOs. The 2′OMe oligomers were incubated in 10% FBSfor periods of 0, 2, 6, or 24 hours, separated by PAGE, stained withmethylene blue, and visualized by transillumination as outlined above.Results are shown in FIGS. 10 and 11.

TABLE 14  Synthetic oligomers employed in FIGS. 10-13 SEQ ID NO: NameSequence 32 2′OMe U C A A C A U C A G U C U G A U A A G C U A 332′OMe PSends U*C*A*A C A U C A G U C U G A U A A G*C*U*A 217 2′OMe 2xC3UxC A A C A U C A G U C U G A U A A G C UxA 38 2′OMe 5′iFQUzC A A C A U C A G U C U G A U A A G C U A 35 2′OMe 5′ + 3′iFQUzC A A C A U C A G U C U G A U A A G C UzA 218 2′OMe 3′FQU C A A C A U C A G U C U G A U A A G C U Az 219 2′OMe 5′iFQ + 3′FQUzC A A C A U C A G U C U G A U A A G C U Az Uppercase = 2′OMe RNA “*” =phosphorothioate linkage “x” = internal C3 spacer (propanediol) “z” =napthyl-azo modifier (FQ)

Degradation of the unmodified 2′OMe RNA oligomer (SEQ ID No. 32)proceeded rapidly with little full length product present after 2 hoursand no full length product present after 6 hours incubation. Theaddition of 3 PS bonds between terminal residues on both the 5′- and3′-ends (SEQ ID No. 33) significantly slowed but did not entirely stopdegradation. By 6 hours, an (n−1)mer species appeared which comprisedaround half of the remaining oligomer by 24 hours. The degradationproduct was identified by mass spectrometry to represent removal of the3′-terminal base, reducing the starting 22-mer oligomer to a 21-merspecies. The oligomer with a C3 spacer (propanediol) placed between theterminal bases on both the 5′- and 3′-ends (SEQ ID No. 217) also showedrapid loss of one base such that 100% of the starting mass was reducedto (n−1)mer by 2 hours incubation; however, no further degradation wasseen at 24 hours, indicating that the C3 spacer element blocked furtherdegradation. The degradation product was identified by mass spectrometryto represent removal of the 3′-terminal base, reducing the starting22-mer oligomer to a 21-mer species. Inserting a single napthyl-azo(iFQ) group between the terminal 5′-residues of the oligomer (SEQ ID No.38) did not significantly affect degradation in serum, and this compoundwas degraded at around the same rate as the unmodified version (SEQ IDNo. 32). Inserting two napthyl-azo (iFQ) groups, one between theterminal 5′-residues and one between the terminal 3′-residues of theoligomer (SEQ ID No. 35) showed loss of one base such that 100% of thestarting mass was reduced to (n−1)mer by 6 hours incubation (slower rateof degradation than was seen using the internal C3 spacer) and nofurther change was seen at 24 hours, indicating that the iFQ modifierblocked further degradation. The degradation product was identified bymass spectrometry to represent removal of the 3′-terminal base, reducingthe starting 22-mer oligomer to a 21-mer species. The oligomer with asingle napthyl-azo (FQ) modifier placed at the 3′-end (not betweenresidues, but instead as a modification of the 3′-hydroxyl of the3′-terminal base) (SEQ ID No. 218) showed no degradation over 24 hours,fully protecting the oligomer from attack by the 3′-exonuclease activitypresent in serum. The oligomer with two napthyl-azo (FQ) modifiers, oneplaced between the 5′-terminal residues and the second placed at the3′-end (SEQ ID No. 219) also showed no degradation over 24 hours.

The degradation studies were next extended to include incubation of thesame set of oligomers in liver cell extracts. While the primary nucleaseactivity in serum is a 3′-exonuclease, cell extracts contain5′-exonuclease, 3′-exonuclease, and endonuclease activities. To functionin live cells or animals, a synthetic antisense oligomer must survivethe nucleases present in both serum (during the delivery phase) and theintracellular environment (during effector phase), and stability in thisenvironment will influence both the magnitude of effect as well as theduration of effect achieved. The 2′OMe oligomers (Table 14) wereincubated in 20% liver cell extracts for periods of 0, 2, 6, or 24hours, separated by PAGE, stained with methylene blue, and visualized bytransillumination as outlined above. Results are shown in FIGS. 12 and13.

The unmodified 2′OMe RNA oligomer (SEQ ID No. 32) showed evidence fordegradation after 2 hours and little full-length product was presentafter 24 hours incubation. The addition of 3 PS bonds between terminalresidues on both the 5′- and 3′-ends (SEQ ID No. 33) significantlyslowed but did not entirely stop degradation. By 24 hours, an (n−1)merspecies appeared which was identified by mass spectrometry to representremoval of the 3′-terminal base, reducing the starting 22-mer oligomerto a 21-mer species. The rate and magnitude of 3′-degradation was lowerin cell extracts than in serum (compare FIGS. 10 and 12). The oligomerwith a C3 spacer (propanediol) placed between the terminal bases on boththe 5′- and 3′-ends (SEQ ID No. 217) also showed loss of one base and100% of the starting mass was reduced to (n−1)mer by 24 hoursincubation. The degradation product was identified by mass spectrometryto represent removal of the 3′-terminal base, reducing the starting22-mer oligomer to a 21-mer species. Inserting a single napthyl-azo(iFQ) group between the terminal 5′-residues of the oligomer (SEQ ID No.38) did not significantly affect degradation in liver cell extracts, andthis compound was degraded at around the same rate as the unmodifiedversion (SEQ ID No. 32). Inserting two napthyl-azo (iFQ) groups, onebetween the terminal 5′-residues and one between the terminal3′-residues of the oligomer (SEQ ID No. 35) showed loss of one base suchthat most of the starting mass has been reduced to (n−1)mer by 24 hoursincubation (again, slower than was seen with incubation in serum). Thedegradation product was identified by mass spectrometry to representremoval of the 3′-terminal base, reducing the starting 22-mer oligomerto a 21-mer species. The oligomer with a single napthyl-azo (FQ)modifier placed at the 3′-end (not between residues, but as amodification of the 3′-hydroxyl of the 3′-terminal base) (SEQ ID No.218) showed some degradation, but >50% of the starting mass remainedintact after 24 hours incubation. This result differs from that seenwhen this same oligomer was incubated in serum where no degradation wasobserved; this oligomer has no 5′-modification and is thus susceptibleto attack by 5′-exonucleases present in cell extracts which are absentin serum. The oligomer with two napthyl-azo (FQ) modifiers, one placedbetween the 5′-terminal residues and the second placed at the 3′-end(SEQ ID No. 219) showed no degradation over 24 hours.

Non-base modifiers such as a C3 spacer or iFQ block degradation byexonucleases when placed between bases, however loss of the externalbase can occur. Exonuclease attack is fully blocked when the modifier ispositioned after the terminal base. 2′OMe RNA oligomers were stable inserum with only 3′-end modification; however, both 5′-end and 3′-endmodifications were needed to achieve full protection in cell extracts.Thus incorporation of modifying groups that confer nuclease resistanceon both ends of the oligomer is preferred.

When synthetic oligonucleotides are delivered to cells in tissue cultureor to animals via IV injection, the compounds are exposed to serum inculture medium or blood for periods lasting from several minutes to manyhours. The serum stability testing shown in FIGS. 10 and 11 showincubation for periods as long as 24 hours, which meets this need. Incontrast, once the synthetic oligonucleotide exits the serum environmentand enters living cells, it will ideally remain intact for many days,during which time the oligomer is active as an anti-miRNA agent, as asplice-switching agent, or as an anti-mRNA ASO. Stability of oligomersin cell extracts for up to 24 hours was demonstrated in FIGS. 12 and 13.

The degradation studies were next extended to include incubation of theset of oligomers shown in Table 15 in mouse liver cell extracts for 4days. Oligomers were incubated in 20% liver cell extracts for either 0or 96 hours, separated by PAGE, stained with methylene blue, andvisualized by transillumination as outlined above. Results are shown inFIG. 14.

TABLE 15  Synthetic oligomers employed in FIG. 14 SEQ ID NO: NameSequence 220 DNA 5′iFQ + 3′FQtzc a a c a t c a g t c t g a t a a g c t az 35 2′OMe 5′ + 3′iFQUzC A A C A U C A G U C U G A U A A G C UzA 218 2′OMe 3′FQU C A A C A U C A G U C U G A U A A G C U Az 219 2′OMe 5′iFQ + 3′FQUzC A A C A U C A G U C U G A U A A G C U Az Uppercase = 2′OMe RNALowercase = DNA “z” = napthyl-azo modifier (FQ)

A DNA oligomer with the napthyl-azo modifier placed between bases at the5′-end and following the terminal base at the 3′-end (SEQ ID No. 220)was completely degraded in cell extracts. In spite of protection fromboth 5′- and 3′-exonuclease attack by the terminal FQ modifying groups,this oligomer remained sensitive to endonucleases present in the cellextract. In contrast, a 2′OMe oligomer with similar end modificationremained fully intact after 4 days incubation in cell extract at 37° C.(SEQ ID No. 219), indicating that, unlike DNA, the 2′OMe modified sugarbackbone protects the compound from endonuclease attack. As expected, a2′OMe oligomer with the napthyl-azo modifier placed between bases at the5′-end and between bases at the 3′-end (SEQ ID No. 35) had the terminal3′-residue removed but was otherwise intact after 4 days incubation incell extract. A 2′OMe oligomer modified only at the 3′-end with thenapthyl-azo group (SEQ ID No. 218) had ˜50% of the input mass surviveincubation for 4 days in cell extract. This finding demonstrates that3′-end modification alone is insufficient to fully protect a 2′OMeoligomer from degradation, presumably from 5′-exonuclease activitypresent in the cell extract.

Thus incorporation of modifying groups that confer exonucleaseresistance on both ends of the oligomer is preferred, and 2′OMe RNA ispreferred over DNA due to its increased resistance to endonucleaseattack. It is anticipated that other 2′-modifications, such as LNA,2′-MOE (2′-O-methoxyethyl), and other 2′-modified sugars, as are wellknown to those with skill in the art, would show similar improvedstability in serum or in cell extracts, and can similarly be used withthe napthyl-azo modifier as taught herein.

EXAMPLE 10

Extending the results from Example 3, this example compares functionalactivity of additional design variants of modified ASOs at reducingmicroRNA activity.

Oligonucleotide Synthesis and Purification.

Oligomers were synthesized using solid phase phosphoramidite chemistry,deprotected and desalted on NAP-5 columns (Amersham Pharmacia Biotech,Piscataway, N.J.) according to routine techniques (Caruthers et al.,1992). The oligomers were purified using reversed-phase high performanceliquid chromatography (RP-HPLC). The purity of each oligomer wasdetermined by capillary electrophoresis (CE) on a Beckman P/ACE MDQsystem (Beckman Coulter, Inc., Fullerton, Calif.). All single-strandoligomers were at least 85% pure. Electrospray-ionization liquidchromatography mass spectrometry (ESI-LCMS) of the oligonucleotides wasconducted using an Oligo HTCS system (Novatia, Princeton, N.J.), whichconsisted of ThermoFinnigan TSQ7000, Xcalibur data system, ProMass dataprocessing software, and Paradigm MS4™ HPLC (Michrom BioResources,Auburn, Calif.). Protocols recommended by the manufacturers werefollowed. Experimental molar masses for all single-strand oligomers werewithin 1.5 g/mol of expected molar mass. These results confirm identityof the oligomers. Table 16 lists the synthetic oligomers used in thisExample, all of which were designed to be perfectly complementary tomiR-21.

TABLE 16  Synthetic oligomers employed in Example 10 (miR-21 AMOs) SEQID NO: Name Sequence 32 2′OMeU C A A C A U C A G U C U G A U A A G C U A 37 2′OMe 3′iFQU C A A C A U C A G U C U G A U A A G C UzA 218 2′OMe 3′FQU C A A C A U C A G U C U G A U A A G C U Az 221 2′OMe 3′iC3U C A A C A U C A G U C U G A U A A G C UxA 38 2′OMe 5′iFQUzC A A C A U C A G U C U G A U A A G C U A 222 2′OMe 5′iC3UxC A A C A U C A G U C U G A U A A G C U A 35 2′OMe 5′iFQ + 3′iFQUzC A A C A U C A G U C U G A U A A G C UzA 217 2′OMe 5′iC3 + 3′iC3UxC A A C A U C A G U C U G A U A A G C UxA 219 2′OMe 5′iFQ + 3′FQUzC A A C A U C A G U C U G A U A A G C U Az 223 2′OMe 5′iC3 + 3′C3UxC A A C A U C A G U C U G A U A A G C U Ax Uppercase = 2′OMe RNA “x” =C3 spacer (propanediol) “z” = napthyl-azo modifier (FQ)

Plasmid Preparation.

The psiCHECK™-2 vector (Promega, Madison, Wis.) was restriction enzymedigested sequentially with XhoI and NotI (New England Biolabs, Ipswitch,Mass.) and purified with a Qiaquick PCR purification column (Qiagen,Valencia, Calif.). A perfect complement hsa-miR-21 binding site wascreated by annealing two synthetic duplexed oligonucleotides (IntegratedDNA Technologies, Coralville, Iowa) and was cloned into the XhoI/NotIsites in the 3′UTR of Renilla luciferase. This miR-21 reporter constructwas sequence verified on a 3130 Genetic Analyzer (AB, Foster City,Calif.). Plasmids were purified using a Plasmid Midiprep Kit (Bio-Rad,Hercules, Calif.) and treated twice for endotoxin removal with theMiraCLEAN Endotoxin Removal Kit (Mirus Corporation, Madison, Wis.).Plasmids were filtered through a 0.2μ filter and quantified bymeasurement of the absorbance at 260 nm using UV spectrophotometry. Thisreporter plasmid having a perfect match miRNA binding site is denoted aspsiCHECK™-2-miR21.

Cell Culture, Transfections, and Luciferase Assays.

HeLa cells were plated in a 100 mm dish in DMEM containing 10% FBS toachieve 90% confluency the next day. The following morning, 5 μg of thepsiCHECK™-2-miR21 plasmid was transfected with Lipofectamine™ 2000(Invitrogen, Carlsbad, Calif.). After 6 hours, cells were washed withPBS, trypsinized, counted, and replated in DMEM with 10% FBS in 48-wellplates to achieve ˜70% confluency the next day. The following morning,the miR-21 AMOs were transfected at various concentrations in triplicatewith 1 μl TriFECTin® (Integrated DNA Technologies) per well in DMEMwithout serum. After 6 hours, the transfection media was removed andreplenished with DMEM containing 10% FBS. The following morning, (48hours after plasmid transfection, 24 hours after miRNA AMO transfection)the cells were analyzed for luciferase luminescence using theDual-Luciferase® Reporter Assay System (Promega, Madison, Wis.) per themanufacturer's instructions. Renilla luciferase was measured as a foldincrease in expression compared to the TriFECTin® reagent-only negativecontrols. Values for Renilla luciferase luminescence were normalized tolevels concurrently measured for firefly luciferase, which is present asa separate expression unit on the same plasmids as an internal control(RLuc/FLuc ratio).

Results.

The RLuc/FLuc ratios obtained from transfections done with the variousAMOs are shown in FIG. 15. In the untreated state, HeLa cells containlarge amounts of miRNA 21 that suppress expression of the RLuc reporter.Any treatment that decreases miR-21 levels leads to an increase in RLucexpression and thus increases the relative RLuc/FLuc ratio (with FLucserving as an internal normalization control for transfectionefficiency).

The unmodified 2′OMe RNA AMO (SEQ ID No. 32) showed essentially noinhibition of miR-21 activity, probably due to rapid nucleasedegradation of this unprotected oligomer during transfection or in theintracellular environment. Protecting either end of the anti-miR-21 AMOfrom exonuclease attack with an internal napthyl-azo modifier improvedpotency when compared with the unmodified 2′OMe AMO. The 2′OMe-3′ iFQ(SEQ ID No. 37, with an internal napthyl-azo modifier between theterminal 3′-residues) show some improvement over the unmodifiedoligomer. Similar results were obtained whether the modifier waspositioned between the terminal bases (SEQ ID No. 37) or at the 3′-end(SEQ ID No. 218, 2′OMe-3′FQ). If an internal C3 (propanediol) spacergroup is positioned between the 3′ residues (2′OMe-3′ iC3, SEQ ID No.221), no functional benefit is observed. Interestingly, thismodification blocks 3′-exonuclease attack, providing significantprotection of the oligomer in both serum and the intracellularenvironment (Example 9), yet does not increase functional potency of theoligomer as an anti-miRNA reagent against this target. 5′-modificationwith the napthyl-azo group placed between terminal residuessignificantly increased potency of miR-21 knockdown (SEQ ID No. 38)whereas a C3 spacer placed at this same position provides no benefit(SEQ ID No. 222). The 2′OMe AMO modified with two napthyl-azo groupswhere one group is placed near the 5′-end between terminal residues andthe other group is placed near the 3′-end between terminal residues (SEQID No. 35) or where one group is placed near the 5′-end between terminalresidues and the other group is placed at the 3′-end (SEQ ID No. 219)both showed very potent inhibition of miR-21 activity and were the mostpotent reagents tested in this survey. In spite of providing equalprotection of the oligomer from degradation (Example 9), the sameoligomers modified with C3 spacers (SEQ ID No. 35 and SEQ ID no. 223)showed no anti-miR-21 activity. Thus nuclease stabilization alone doesnot account for the large improvement in AMO activity seen with use ofthe napthyl-azo modifier. Increased binding affinity likely is a secondkey element to the benefit obtained from use of this modificationstrategy.

The melting temperatures, T_(m), of the AMOs described above weremeasured using the same methods described in Example 8 and are shown inTable 17 below. Synthetic AMO oligonucleotides were annealed to asynthetic RNA complement (mature miR-21 RNA sequence, SEQ ID No. 114, 5′phos-UAGCUUAUCAGACUGAUGUUGA 3′). Measurements were done at 2 μM duplexconcentration in 150 mM NaCl to approximate intracellular ionconcentration. Measurements were made on both the melt and re-annealphase and repeated 3 times, so the values shown represent the average of6 T_(m) measurements. The ΔT_(m) is calculated as the difference betweenthe modified AMO variant and the unmodified version (SEQ ID No. 32).Note that the accuracy of T_(m) measurement is around +/−0.5° C. so thatthe T_(m) values for a given duplex reported in this experiment may varyslightly from that reported in earlier examples for the same duplex.

TABLE 17  T_(m) of synthetic miR-21 AMOs in 150 mM NaCl SEQ ID NO: NameSequence T_(m)° C. ΔT_(m)° C. 32 2′OMeU C A A C A U C A G U C U G A U A A G C U A 72.0 — 37 2′OMe 3′iFQU C A A C A U C A G U C U G A U A A G C UzA 72.4 +0.4 218 2′OMe 3′FQU C A A C A U C A G U C U G A U A A G C U Az 72.6 +0.6 221 2′OMe 3′iC3U C A A C A U C A G U C U G A U A A G C UxA 72.1 +0.1 38 2′OMe 5′iFQUzC A A C A U C A G U C U G A U A A G C U A 75.4 +3.4 222 2′OMe 5′iC3UxC A A C A U C A G U C U G A U A A G C U A 71.6 −0.4 35 2′OMeUzC A A C A U C A G U C U G A U A A G C UzA 75.6 +3.6 5′iFQ + 3′iFQ 2172′OMe UxC A A C A U C A G U C U G A U A A G C UxA 71.1 −0.9 5′iC3 +3′iC3 219 2′OMe UzC A A C A U C A G U C U G A U A A G C U Az 76.0 +4.05′iFQ + 3′FQ 223 2′OMe UxC A A C A U C A G U C U G A U A A G C U Ax 72.6+0.6 5′iC3 + 3′C3 Uppercase = 2′OMe RNA “x” = C3 spacer (propanediol)“z” = napthyl-azo modifier (FQ)

As a general rule, the effects of the C3 spacer on T_(m) was neutral orslightly destabilizing (greatest drop seen was −0.9° C. for thedual-modified SEQ ID No. 217) while the napthyl-azo modifier wasslightly to significantly stabilizing (greatest gain seen was +4.0° C.for SEQ ID No. 219). The relative potency of the various miR-21 AMOscorrelated with binding affinity (T_(m)). All variations in potencyobserved between compounds could be explained by relative contributionsof improvements in binding affinity and nuclease stability between thedifferent modification patterns studied. The AMO having 2′OMe bases withan FQ modification placed near each end (“2′OMe 5′ iFQ+3′ iFQ”, SEQ IDNo. 35) and that with an FQ modification placed near the 5′-end and atthe 3′-end (“2′OMe 5′ iFQ+3′FQ”, SEQ ID No. 219) both showed anexcellent balance of nuclease stability with increased T_(m). Thevariant with the napthyl-azo modifier located between terminal bases atthe 3′-end (SEQ ID No. 35), however, is at risk for loss of the terminalresidue by exonuclease attack whereas the having the modifier at the3′-end protects the terminal residue (SEQ ID No. 219) (see Example 9)and therefore use of this design may be preferred in some settings.

EXAMPLE 11

Extending the results from Example 10, this example compares functionalactivity of additional design variants of modified ASOs at reducingmicroRNA activity by optimizing 3′-end structure.

Oligonucleotide Synthesis and Purification.

Oligomers were synthesized using solid phase phosphoramidite chemistry,deprotected and desalted on NAP-5 columns (Amersham Pharmacia Biotech,Piscataway, N.J.) according to routine techniques (Caruthers et al.,1992). The oligomers were purified using reversed-phase high performanceliquid chromatography (RP-HPLC). The purity of each oligomer wasdetermined by capillary electrophoresis (CE) on a Beckman P/ACE MDQsystem (Beckman Coulter, Inc., Fullerton, Calif.). All single-strandoligomers were at least 85% pure. Electrospray-ionization liquidchromatography mass spectrometry (ESI-LCMS) of the oligonucleotides wasconducted using an Oligo HTCS system (Novatia, Princeton, N.J.), whichconsisted of ThermoFinnigan TSQ7000, Xcalibur data system, ProMass dataprocessing software, and Paradigm MS4™ HPLC (Michrom BioResources,Auburn, Calif.). Protocols recommended by the manufacturers werefollowed. Experimental molar masses for all single-strand oligomers werewithin 1.5 g/mol of expected molar mass. These results confirm identityof the oligomers. Table 18 lists the synthetic oligomers used in thisExample, all of which were designed to be perfectly complementary tomiR-21.

TABLE 18  Synthetic oligomers employed in Example 11 (miR-21 AMOs) SEQID NO: Name Sequence 224 2′OMe 22 3′C3U C A A C A U C A G U C U G A U A A G CU Ax 225 2′OMe 22 3′FQU C A A C A U C A G U C U G A U A A G C U Az 226 2′OMe 22 3′iFQU C A A C A U C A G U C U G A U A A G C UzA 227 2′OMe 21 3′FQU C A A C A U C A G U C U G A U A A G C Uz 228 2′OMe 21 3′iFQU C A A C A U C A G U C U G A U A A G CzU 229 2′OMe 20 3′FQU C A A C A U C A G U C U G A U A A G Cz   230 2′OMe 20 3′iFQU C A A C A U C A G U C U G A U A A GzC 231 2′OMe 19 3′FQU C A A C A U C A G U C U G A U A A Gz Uppercase = 2′OMe RNA “x = C3spacer (propanediol) “z” = napthyl-azo modifier (FQ)

Plasmid Preparation.

The psiCHECK™-2 vector (Promega, Madison, Wis.) was restriction enzymedigested sequentially with XhoI and NotI (New England Biolabs, Ipswitch,Mass.) and purified with a Qiaquick PCR purification column (Qiagen,Valencia, Calif.). A perfect complement hsa-miR-21 binding site wascreated by annealing two synthetic duplexed oligonucleotides (IntegratedDNA Technologies, Coralville, Iowa) and was cloned into the XhoI/NotIsites in the 3′UTR of Renilla luciferase. This miR-21 reporter constructwas sequence verified on a 3130 Genetic Analyzer (AB, Foster City,Calif.). Plasmids were purified using a Plasmid Midiprep Kit (Bio-Rad,Hercules, Calif.) and treated twice for endotoxin removal with theMiraCLEAN Endotoxin Removal Kit (Minis Corporation, Madison, Wis.).Plasmids were filtered through a 0.2μ filter and quantified bymeasurement of the absorbance at 260 nm using UV spectrophotometry. Thisreporter plasmid having a perfect match miRNA binding site is denoted aspsiCHECK™-2-miR21.

Cell Culture, Transfections, and Luciferase Assays.

HeLa cells were plated in a 100 mm dish in DMEM containing 10% FBS toachieve 90% confluency the next day. The following morning, 5 μg of thepsiCHECK™-2-miR21 plasmid was transfected with Lipofectamine™ 2000(Invitrogen, Carlsbad, Calif.). After 6 hours, cells were washed withPBS, trypsinized, counted, and replated in DMEM with 10% FBS in 48-wellplates to achieve ˜70% confluency the next day. The following morning,the miR-21 AMOs were transfected at various concentrations in triplicatewith 1 μl TriFECTin® (Integrated DNA Technologies) per well in DMEMwithout serum. After 6 hours, the transfection media was removed andreplenished with DMEM containing 10% FBS. The following morning, (48hours after plasmid transfection, 24 hours after miRNA AMO transfection)the cells were analyzed for luciferase luminescence using theDual-Luciferase® Reporter Assay System (Promega, Madison, Wis.) per themanufacturer's instructions. Renilla luciferase was measured as a foldincrease in expression compared to the TriFECTin® reagent-only negativecontrols. Values for Renilla luciferase luminescence were normalized tolevels concurrently measured for firefly luciferase, which is present asa separate expression unit on the same plasmids as an internal control(RLuc/FLuc ratio).

Results.

The RLuc/FLuc ratios obtained from transfections done with the variousAMOs are shown in FIG. 16. In the untreated state, HeLa cells containlarge amounts of miRNA 21 that suppress expression of the RLuc reporter.Any treatment that decreases miR-21 levels leads to an increase in RLucexpression and thus increases the relative RLuc/FLuc ratio (with FLucserving as an internal normalization control for transfectionefficiency).

This study examines the effects that shortening the 3′-end of theanti-miRNA ASO (AMO) has on potency and on the precise placement of thenapthyl-azo modifier. The 3′-end of the AMO hybridizes to the 5′-end ofthe miRNA, which is known as the “seed region” and is critical for miRNAactivity. All AMOs in the present example had an unmodified 5′-end. The3′-end was protected with a 3′-modifier (such as an iFQ napthyl-azogroup or an iC3 spacer), which blocks attack of the 3′-end byexonuclease activity (see Example 9). Alternatively an iFQ modifier wasplaced between the 3′-terminal base and the next base. This placementpermits removal of the 3′-terminal base by exonuclease activity,shortening the AMO by one base during transfection via exposure to serumand/or cellular nucleases, however thermodynamic benefit and thereforepotency increases will still be imparted by the iFQ group.

The 2′OMe 22 3′C3 (SEQ ID No. 224) serves as a control for the potencyof the full length 22mer AMO without any group to enhance thermodynamicstability and uses a 3′-C3 end block to prevent exonuclease attack. Thisspecies will remain 22mer after incubation in serum and cell extract(see Example 9). The 2′OMe 22 3′FQ AMO (SEQ ID No. 225) shows theincrease in potency gained by using the napthyl-azo modifier instead ofthe C3 spacer. It also remains 22mer length after incubation in serum orcell extract. Interestingly, the most potent designs were those whichsurvive within cells as an (n−1)mer species with a single basetruncation from the 3′-end (for miR-21, this is a 21mer species). The2′OMe 22 3′ iFQ (SEQ ID No. 226) will degrade to a 21mer in serum orcell extracts while the 2′OMe 21 3′FQ (SEQ ID No. 227) is protected fromloss of the terminal base; it was synthesized as a 21mer and remains a21mer. Both of these designs showed the highest potency, indicating thatthis 3′-end design for the AMO may be preferred, at least in certainsequence contexts. Further shortening of the AMO to 20mer length, 2′OMe21 3′ iFQ (SEQ ID No. 228) or 2′OMe 20 3′FQ (SEQ ID No. 229) showedreduced activity and yet additional shortening of the AMO to 19merlength, 2′OMe 20 3′ iFQ (SEQ ID No. 230) or 2′OMe 19 3′FQ (SEQ ID No.231) had no detectable anti-miR-21 activity in this assay system.

EXAMPLE 12

This example demonstrates improved functional activity of terminalnapthyl-azo-modified oligomers at reducing cellular mRNA levels whenincorporated into RNase H active ASOs as compared to other relatedcompounds.

Example 5 demonstrates improved function of ASOs containing internalmodification with a T_(m)-enhancing modification placed near the ends ofthe oligonucleotide at the penultimate position, between the last andnext to last nucleotide. The present example demonstrates even moreimproved function when the T_(m)-enhancing modifier is placed at the 5′-and 3′-ends of the oligonucleotide.

Oligonucleotides antisense in orientation to cellular messenger RNAs(mRNAs) will hybridize to the mRNA and form an RNA/DNA heteroduplex,which is a substrate for cellular RNase H. Degradation by RNase H leadsto a cut site in the mRNA and subsequently to total degradation of thatRNA species, thereby functionally lowering effective expression of thetargeted transcript and the protein it encodes. ASOs of this typerequire a domain containing at least 4 bases of DNA to be a substratefor RNase H, and maximal activity is not seen until 8-10 DNA bases arepresent. ASOs must be chemically modified to resist degradation by serumand cellular nucleases. Phosphorothioate (PS) modification of theinternucleotide linkages is compatible with RNase H activation, howevermost other nuclease resistant modifications prevent RNase H activity,including all 2′-modifications, such as 2′OMe RNA, LNA, MOE, etc. The PSmodification lowers binding affinity (T_(m)). In general, modificationsthat lower T_(m) decrease potency while modifications that increaseT_(m) improve potency. One strategy to improve potency of ASOs is toemploy a chimeric design where a low T_(m), RNase H activating domainmade of PS-modified DNA is flanked by end domains that contain2′-modified sugars which confer high binding affinity but are not RNaseH activating (“gapmer” design). One commonly employed strategy is toplace five 2′-modified residues at the 5′-end, ten PS-modified DNAresidues in the middle, and five 2′-modified residues at the 3′-end ofthe ASO (so called “5-10-5” gapmer design). Shorter ASOs, such as thosethat contain three 2′-modified residues at the 5′-end, ten PS-modifiedDNA residues in the middle, and three 2′-modified residues at the 3′-end(“3-10-3” gapmer design) will have lower T_(m) than the longer “5-10-5”design and will have lower potency when using facilitated deliverymethods (such as cationic lipid mediated transfection) than the “5-10-5”ASOs but can show higher potency when administered in vivo using nakedIV injection. In general, short oligonucleotides (around 16 residues, orpreferable 12-14 residues, or less) enter mammalian cells better whenadministered without the aid of a delivery tool than longeroligonucleotides (Straarup et al., 2010, Nucleic Acids Res.38(20):7100-7111. A modification that confers nuclease resistance,increases binding affinity, and does not impair the reagent's ability toactivate RNase H may increase the potency of the shorter ASOs to comparewith longer ASOs yet might retain the improved unassisted deliverycharacteristics of the shorter compounds. The present exampledemonstrates the utility of placing a T_(m)-enhancing modification, inthis case the napthyl-azo modifier(N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine) at the 3′- and5′-ends of ASOs to improve the nuclease stability, increase bindingaffinity and enhance potency as gene knockdown reagents.

Oligonucleotide Synthesis and Purification.

DNA, 2′OMe RNA, and LNA containing oligonucleotides were synthesizedusing solid phase phosphoramidite chemistry, deprotected and desalted onNAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.) accordingto routine techniques (Caruthers et al., 1992). Electrospray-ionizationliquid chromatography mass spectrometry (ESI-LCMS) of theoligonucleotides was conducted using an Oligo HTCS system (Novatia,Princeton, N.J.), which consisted of ThermoFinnigan TSQ7000, Xcaliburdata system, ProMass data processing software, and Paradigm MS4™ HPLC(Michrom BioResources, Auburn, Calif.). Protocols recommended by themanufacturers were followed. Experimental molar masses for allsingle-strand oligomers were within 1.5 g/mol of expected molar mass.These results confirm identity of the oligomers.

TABLE 19  Synthetic oligomers employed in Example 12 (anti-HPRT ASOs)SEQ ID NO: Name Sequence 232 HPRT1 3-10-3 LNA PSA*G*G*a*c*t*c*c*a*g*a*t*g*T*T*T 233 HPRT1 3-10-3 LNA PS A _(z)G*G*a*c*t*c*c*a*g*a*t*g*T*T _(z) T 2x iFQ 234 HPRT1 3-10-3 LNA PS _(z)A*G*G*a*c*t*c*c*a*g*a*t*g*T*T*T _(z) 2x FQ 235 HPRT1 3-10-3 QLNA gapPS _(z) AGG*a*c*t*c*c*a*g*a*t*g*TTT _(z) 2x FQ 236 HPRT1 3-10-3 PSA*G*G*a*c*t*c*c*a*g*a*t*g*U*U*U 237 HPRT1 3-10-3 PSA_(z)G*G*a*c*t*c*c*a*g*a*t*g*U*U_(z)U 2x iFQ 238 HPRT1 3-10-3 PS_(z)A*G*G*a*c*t*c*c*a*g*a*t*g*U*U*U_(z) 2x FQ 239 HPRT1 3-10-3 gapPS_(z)AGG*a*c*t*c*c*a*g*a*t*g*UUU_(z) 2x FQ Uppercase = 2′OMe RNALowercase = DNA Uppercase with underscore =LNA “*” = phosphorothioatelinkage “_(z)” = napthyl-azo modifier (iFQ, FQ)

HeLa Cell Culture, Transfections, and RT-qPCR Methods.

HeLa cells were reverse transfected into 96-well plates in Dulbecco'sModified Eagle Medium containing 10% fetal bovine serum (ATCC, Manassas,Va.) using RNAiMAX® (Life Technologies, Carlsbad, Calif.) at aconcentration of 2% (1 μL per 50 μL OptiMEM® I) (Life Technologies) withASOs at the indicated concentrations. All transfections were performedin triplicate. RNA was prepared 24 hours after transfection using theSV96 Total RNA Isolation Kit (Promega, Madison, Wis.). cDNA wassynthesized using 150 ng total RNA with SuperScript™-II ReverseTranscriptase (Life Technologies) per the manufacturer's instructionsusing both random hexamer and oligo-dT priming. Quantitative real-timePCR was performed using 10 ng cDNA per 10 μL reaction with Immolase™ DNAPolymerase (Bioline, Randolph, Mass.), 500 nM primers, and 250 nM probe.Hypoxanthine phosphoribosyltransferase 1 (HPRT1) (GenBank Acc. No.NM_000194) specific primers and probe were:

HPRT-For (SEQ ID NO: 79) 5′ GACTTTGCTTTCCTTGGTCAGGCA, HPRT-Rev(SEQ ID NO: 80) 5′ GGCTTATATCCAACACTTCGTGGG, HPRT-P (SEQ ID NO: 240) 5′FAM-ATGGTCAAG/ZEN/GTCGCAAGCTTGCTGGT-IBFQ.Samples were normalized to levels of an internal control gene, humansplicing factor, arginine/serine-rich 9 (SFRS9) (GenBank Acc. No.NM_003769), in a multiplexed reaction using primers and probe:

SFRS9-For (SEQ ID NO: 241) 5′ TGTGCAGAAGGATGGAGT, SFRS9-Rev(SEQ ID NO: 242) 5′ CTGGTGCTTCTCTCAGGATA, SFRS9-P (SEQ ID NO: 243) 5′HEX-TGGAATATG/ZEN/CCCTGCGTAAACTGGA-IBFQ.Cycling conditions employed were: 95° C. for 10 minutes followed by 40cycles of 2-step PCR with 95° C. for 15 seconds and 60° C. for 1 minute.PCR and fluorescence measurements were done using an ABI Prism™ 7900Sequence Detector (Life Technologies). All reactions were performed intriplicate. Copy number standards were multiplexed using linearizedcloned amplicons for both the HPRT and SFRS9 assays. Unknowns wereextrapolated against standards to establish absolute quantitativemeasurements.

Results.

ASOs were transfected into HeLa cells at 3 nM, 10 nM, and 30 nMconcentrations. RNA was prepared 24 hours post transfection, convertedto cDNA, and HPRT expression levels were measured using qPCR. Resultsare shown in FIG. 17. The LNA-PS gapmer (“HPRT1 3-10-3 LNA PS”, SEQ IDNo. 232) was able to suppress HPRT expression by 85% when used at 30 nMand 65% at 10 nM concentrations. With the addition of theT_(m)-enhancing modifications placed near the ends of theoligonucleotide at the penultimate position, between the last and nextto last nucleotide, the LNA-PS gapmer (“HPRT1 3-10-3 LNA PS 2× iFQ”, SEQID No. 233) showed improved potency with 95% knockdown of HPRT achievedwhen used at 30 nM and 90% knockdown at 10 nM concentrations. Withaddition of the T_(m)-enhancing modification at the 5′- and 3′-ends ofthe oligonucleotide, the LNA-PS gapmer (“HPRT1 3-10-3 LNA PS 2× FQ”, SEQID No. 234) showed further increases in potency with over 95% knockdownof HPRT when used at 30 nM and over 90% knockdown when used at 10 nMconcentrations. This demonstrates the utility of the T_(m)-enhancingmodifier when used either near or at the 3′- and 5′-ends of the ASO.

For the 2′OMe versions of the short 3-10-3 design ASOs studied in thepresent example, the parent 2′OMe-PS gapmer (“HPRT1 3-10-3 PS”, SEQ IDNo. 236) was unable to reduce HPRT mRNA levels at the doses studied.This negative result was expected, as the 2′OMe modification does notconfer a sufficient increase in binding affinity for this chemicalcomposition to function as a short 3-10-3 design whereas the LNAmodification provides a sufficient increase in binding affinity to beeffective. In longer 5-10-5 designs, the 2′OMe gapmers can suppress HPRTmRNA levels (see Example 5, FIGS. 5 and 6). However, addition of the newnon-nucleotide modifier to the 2′OMe 3-10-3 gapmer ASOs increasespotency and a significant reduction in HPRT mRNA levels was observedusing this new class of reagents. The 2′OMe gapmer with addition of theT_(m)-enhancing modifications placed near the ends of theoligonucleotide at the penultimate position, between the last and nextto last nucleotide (“HPRT1 3-10-3 PS 2× iFQ”, SEQ ID No. 237), showedimproved potency with 85% knockdown of HPRT achieved when used at 30 nMand 65% knockdown at 10 nM concentrations. This efficacy was similar tothat seen using the LNA-PS 3-10-3 gapmer (SEQ ID No. 232) With additionof the T_(m)-enhancing modification at the 5′- and 3′-ends of theoligonucleotide, the 2′OMe-PS gapmer (“HPRT1 3-10-3 PS 2× FQ”, SEQ IDNo. 238) showed further increases in potency with over 90% knockdown ofHPRT when used at 30 nM and almost 80% knockdown when used at 10 nMconcentrations. These data further demonstrate the utility of theT_(m)-enhancing modifier when used either near or at the 3′- and 5′-endsof the ASO.

The PS modification confers nuclease resistance but also lowers bindingaffinity. The variant of the LNA-PS gapmer having a terminalT_(m)-enhancing modifier but with phosphodiester linkages between the2′-modified LNA residues (“HPRT1 3-10-3 LNA gapPS 2× FQ”, SEQ ID No.235) showed even greater increases in potency, with 90% knockdown ofHPRT mRNA seen using a 3 nM dose. Therefore use of the terminalT_(m)-enhancing modifier, which both increases binding affinity andblocks exonuclease attack, permits reduction in the amount of PSmodification needed in the ASO, limited to the DNA segment in the middledomain of the gapmer. Reduction in PS content can lead to increases inpotency and decreases in toxicity.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. An antisense oligonucleotide comprising at leastone modification that is incorporated at the terminal end or between twonucleotides of the antisense oligonucleotide, wherein the modificationincreases binding affinity and nuclease resistance of the antisenseoligonucleotide, and wherein the modification is a napthyl-azo compound.2. The antisense oligonucleotide of claim 1, wherein the modification islocated at the 5′-end of the antisense oligonucleotide.
 3. The antisenseoligonucleotide of claim 1, wherein the modification is located at the3′-end of the antisense oligonucleotide.
 4. The antisenseoligonucleotide of claim 1, wherein modifications are located at boththe 5′- and 3′-ends of the antisense oligonucleotide.
 5. The antisenseoligonucleotide of claim 1, wherein the modification is inserted betweennucleotides and is located near the 3′-end of the antisenseoligonucleotide.
 6. The antisense oligonucleotide of claim 1, whereinthe modification is inserted between nucleotides and is located near the5′-end of the antisense oligonucleotide.
 7. The antisenseoligonucleotide of claim 1, wherein modifications are inserted betweennucleotides and are located near the 3′-end and the 5′-end of theantisense oligonucleotide.
 8. The antisense oligonucleotide of claim 1,wherein a modification is located at the 5′-end and a modification isinserted between nucleotides near the 3′-end of the antisenseoligonucleotide.
 9. The antisense oligonucleotide of claim 1, wherein amodification is located at the 3′-end and a modification is insertedbetween nucleotides near the 5′-end of the antisense oligonucleotide.10. The antisense oligonucleotide of claim 1, wherein the modificationhas the structure:

wherein the linking groups L₁ and L₂ positioning the modification at aninternal position of the oligonucleotide are independently an alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R₁, R₂, R₄,R₅ are independently a hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl,substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl,alkylaryl, alkoxy, an electron withdrawing group, or an electrondonating group; R₆, R₇, R₉-R₁₂ are independently a hydrogen, alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron withdrawinggroup, or an electron donating group; R₈ is a hydrogen, alkyl, alkynyl,alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substitutedaryl, cycloalkyl, alkylaryl, alkoxy, or an electron withdrawing group;and X is a nitrogen or carbon atom, wherein if X is a carbon atom, thefourth substituent attached to the carbon atom can be hydrogen or aC1-C8 alkyl group.
 11. The antisense oligonucleotide of claim 10,wherein R₈ is NO₂.
 12. The antisense oligonucleotide of claim 1, whereinthe modification has the structure:


13. The antisense oligonucleotide of claim 1, wherein the antisenseoligonucleotide further comprises at least one 2′-O-methyl RNA residue.14. The antisense oligonucleotide of claim 13, further comprising atleast one napthyl-azo compound modification that is incorporated at theterminal end of the antisense oligonucleotide, wherein the antisenseoligonucleotide targets miRNA.
 15. The antisense oligonucleotide ofclaim 14, wherein the antisense oligonucleotide is the same length asthe miRNA target.
 16. The antisense oligonucleotide of claim 14, whereinthe antisense oligonucleotide is one base shorter than the miRNA target.17. The antisense oligonucleotide of claim 1, wherein the antisenseoligonucleotide further comprises one or more phosphorothioateinternucleotide linkages.
 18. The antisense oligonucleotide of claim 1,wherein each nucleotide of the antisense oligonucleotide isphosphorothioate modified.
 19. The antisense oligonucleotide claim 18,further comprising a chemical modification, wherein the chemicalmodification comprises 2′OMe RNA, 2′F RNA, or LNA bases.
 20. Theantisense oligonucleotide of claim 1, wherein the antisenseoligonucleotide comprises a region of bases linked throughphosphodiester bonds, wherein the region is flanked at one or both endsby regions containing phosphorothioate linkages.
 21. The antisenseoligonucleotide of claim 1, wherein the antisense oligonucleotidecomprises a region of bases linked through phosphorothioate bonds,wherein the region is flanked at one or both ends by regions containingphosphodiester linkages.
 22. An oligonucleotide having the structure:5′-X₁-Z_(n)-X₂-X₃-X₄-Z_(n)-X₅-3′ wherein X₁ and X₅ are independently 0-3nucleotides wherein the internucleotide linkages are optionallyphosphorothioate; Z is a napthyl-azo compound; at least one n is 1, andthe other n is 0 or 1; X₂ and X₄ are independently 1-5 nucleotideswherein the internucleotide linkages are optionally phosphorothioate;and X₃ is 10-25 nucleotides, wherein the linkages are optionallyphosphorothiaote.
 23. An oligonucleotide complementary to a target mRNAcomprising: (a) a modified 3′-terminal internucleotide phosphodiesterlinkage, which modified 3′-terminal internucleotide phosphodiesterlinkage is resistant to 3′ to 5′ exonuclease degradation; (b)modifications on the 3′-terminus and the 5′-terminus of theoligonucleotide, wherein the modifications increase binding affinity ofthe oligonucleotide to the target mRNA; (c) one or more additionalmodifications, which additional modification(s) facilitate(s)intracellular transport of said oligodeoxynucleotide; and (d) acontinuous stretch of at least five nucleotide residues having fourinternucleotide phosphodiester linkages which are unmodified, whereinsaid oligodeoxynucleotide, when mixed with an RNA molecule for which ithas complementarity under conditions in which an RNaseH is active,hybridizes to the RNA and forms a substrate that can be cleaved by theRNase H; and wherein at least one of the modifications comprises anapthyl-azo compound.
 24. The antisense oligonucleotide of claim 13,wherein the antisense oligonucleotide contains one or more DNA moleculesand wherein each 2′OMe and nucleotide is connected by a phosphorothioategroup.
 25. The antisense oligonucleotide of claim 24, additionallycomprising at least one napthyl-azo compound modification that isincorporated at the terminal end of the antisense oligonucleotide,wherein the modification increases stability of the antisenseoligonucleotide.
 26. The antisense oligonucleotide of claim 24, whereinthe length of each 2′OMe portion is 2-5 nucleotides.
 27. The antisenseoligonucleotide of claim 26, wherein the length of each 2′OMe portion is5 nucleotides.
 28. The antisense oligonucleotide of claim 27 having theformula:zM*M*M*M*M*D*D*D*D*D*D*D*D*D*D*M*M*M*M*Mz wherein z is a napthyl-azocompound, M is 2′O methyl RNA, D is DNA and * is a phosphorothioatelinkage.
 29. The antisense oligonucleotide of claim 13 having theformula:zMMMMM*D*D*D*D*D*D*D*D*D*D*MMMMMz wherein z is a napthyl-azo compound, Mis 2′O methyl RNA, D is DNA and * is a phosphorothioate linkage.
 30. Theantisense oligonucleotide of claim 1, wherein the antisenseoligonucleotide contains one or more LNA molecules and wherein the LNAmolecules optionally contain phosphorothioate linkage groups.
 31. Theantisense oligonucleotide of claim 30, wherein the modification is anapthyl-azo compound that is incorporated at the terminal end of theantisense oligonucleotide, wherein the modification increases stabilityof the antisense oligonucleotide.
 32. The antisense oligonucleotide ofclaim 31, wherein the length of each LNA portion is 2-5 nucleotides. 33.The antisense oligonucleotide of claim 32, wherein the length of eachLNA portion is 5 nucleotides.
 34. The antisense oligonucleotide of claim27 having the formula:zL*L*L*L*L*D*D*D*D*D*D*D*D*D*D*L*L*L*L*Lz wherein z is a napthyl-azocompound, D is DNA, L is LNA, and * is a phosphorothioate linkage. 35.The antisense oligonucleotide of claim 31, wherein the antisenseoligonucleotide has a reduced phosphorothioate group content.
 36. Theantisense oligonucleotide of claim 27 having the formula:zLLLLL*D*D*D*D*D*D*D*D*D*D*LLLLLz wherein z is a napthyl-azo compound, Dis DNA, L is LNA, and * is a phosphorothioate linkage.
 37. The antisenseoligonucleotide of claim 1 having the formula:z_(n)M_(m)*D_(y)*M_(m)*Mz_(n) where M=2′OMe or LNA, D=DNA or a DNAanalogue and z=a napthyl-azo compound, wherein at least one n=1 and asecond n=0 or 1, where m=2-5 and where y=7-12.
 38. An anti-miRNAoligonucleotide (AMO) comprising, (a) at least one 2′-O-methyl RNA(2′OMe), and (b) at least one napthyl-azo compound modification that isincorporated at the terminal end of the AMO, wherein the modificationincreases stability of the AMO.
 39. An RNase H antisense oligonucleotide(ASO) comprising, at least one napthyl-azo compound modification that isincorporated at the terminal end of the ASO, wherein each nucleotide isconnected by a phosphorothioate group (PS) and wherein the modificationincreases stability of the ASO.
 40. The antisense oligonucleotide ofclaim 13 having the formula:MzMMMMMMMMMMMMMMMMMMMMz wherein z is a napthyl-azo compound and M is 2′Omethyl RNA.
 41. The antisense oligonucleotide of claim 40, wherein thenapthyl-azo compound has the formula:


42. The antisense oligonucleotide of claim 40, wherein allinternucleotide linkages are phosphodiester internucleotide linkages.